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Class 
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Copyright^ . 



COPYRIGHT DEPOSIT. 



A MANUAL 



CLINICAL DIAGNOSIS 

BY MEANS OF MICROSCOPICAL AND 
CHEMICAL METHODS, 



STUDENTS, HOSPITAL PHYSICIANS, AND PRACTITIONERS. 



BY 



CHAELES E. SIMON, M.D., 



Of Baltimore. Md. 



FIFTH EDITION, THOROUGHLY REVISED AND ENLARGED. 



ILLUSTRATED WITH 150 ENGRAVINGS AND 22 PLATES IN COLORS. 







LEA BROTHERS & CO., 

PHILADELPHIA AND NEW YOEK. 
1904, 






UBRaKY ui CONGRESS 
Two Copies Received 

MAR 16 1904 

. Copyrigm 5«try 
CLASS ft- Xko. No, 

t:cW 8 



Entered according to Act of Congress, in the year 1904, by 

LEA BROTHERS & CO., 

In the Office of the Librarian of Congress, at Washington. All rights reserved. 



WESTCOTT &. THOMSON. 
ELECTROTYPERS, PHILADA. 



TO 



MY WIFE, 



WHO HAS SO FAITHFULLY AIDED IN ITS PEEPAEATION, 

THIS EDITION ALSO 

is 

' AFFECTIONATELY DEDICATED. 



PREFACE TO THE FIFTH EDITION. 



The demand for a new edition has been construed by the author 
not only as an expression of professional favor, but also and more 
particularly as still another opportunity of keeping the book abreast 
of a most active and important branch of medicine. Exact methods 
of diagnosis are of comparatively recent development, and as they 
necessarily underlie successful therapeusis, they should be a part 
of the equipment of every physician. They are not recondite or 
abstruse, but on the contrary are susceptible of plain statement and 
direct application. Such has been the object of this work from its 
inception, namely, to simplify the physician's work and to increase 
its efficiency by enabling him to eliminate doubt from his diagnosis. 
The book has been adapted to the needs of students as well as of 
graduates, in view of the fact that the subject is already included 
in the curricula of many colleges, and is destined to be required in 
all. The author's endeavor has been to state the best methods 
clearly and simply, with all necessary instructions, and to render 
the book as modern and practical as possible. 

Besides a careful revision, this edition embodies much new matter 
which has appeared in the literature of the past two years. The 
chapter on the blood has been almost entirely rewritten, and has 
been enlarged by sixty pages. Special pains have been taken 
with the chapter on technique. A section dealing with the nature 
of anilin dyes and the principles of staining has been introduced, 
which it is hoped will render this portion of the book more inter- 
esting to the clinical laboratory worker, and will serve as a guide 
to further investigation. For convenience of reference, the subject 
of leucocytosis has been rearranged in such manner that hyper- 
leucocytosis and hypoleucocytosis are separately considered in con- 
nection with the different varieties of leucocytes. A new section 
deals with the kryoscopic examination of the blood. The bac- 
teriology and parasitology of the blood have been enlarged, with 



Vl PREFACE TO THE FIFTH EDITION. 

sections on paratyphoid fever, gonococcus septicaemia, bubonic 
plague, trypanosomiasis, and spotted fever. Material changes and 
additions have furthermore been made in the chapters on the feces, 
the sputum, the urine, and on transudates and exudates, and minor 
alterations occur throughout the book. Illustrations have been 
added wherever they appeared necessary to elucidate the text. 

To the profession at large I am indebted for the kind reception 
which has been accorded to the Diagnosis. To Messrs. Lea 
Brothers & Co. I wish to express my appreciation of their many 
acts of courtesy and liberality. The six new colored plates (Nos. I., 
II., III., IV., VI., and XXI.) I owe to my wife, and thank her 
at this place also for the many hours of patient study which she has 
spent in my laboratory for the purpose of familiarizing herself with 
blood morphology more particularly, so as to be able to produce 
illustrations which should be more nearly true to nature than any 
that have hitherto been prepared. To what extent she has suc- 
ceeded I leave to the laboratory student to judge. 

Charles E. Simon. 

1902 Madison Ave., Baltimore, Md., 
January, 1904. 



PREFACE TO THE FIRST EDITION. 



It is curious to note that, notwithstanding the great importance 
of clinical chemistry and microscopy, but little attention is paid to 
these subjects, either by hospital physicians or by those engaged in 
general practice. This lack of interest is referable primarily to the 
fact that a systematic study of these branches has hitherto been 
greatly neglected, not only in American medical schools, but also in 
those of Europe. 

It is no rarity to hear physicians in general practice claim that 
they are too busy to conduct careful examinations of the urine, 
sputum, blood, gastric juice, etc. Would it not be reasonable to 
suppose, however, that a physician who is overwhelmed with work 
to such an extent that he cannot find the time to make use of aids 
in diagnosis which are quite as important as the stethoscope, the 
laryngoscope, or the ophthalmoscope, would be in a position to 
employ an assistant in his laboratory ? The younger practitioner 
is certainly not placed in such a dilemma, and it is a fair assump- 
tion that he could successfully compete with his more experienced 
colleague, in matters of diagnosis at least, were he to familiarize 
himself sufficiently with laboratory methods of diagnosis. 

The time is at hand when the practice of medicine is becoming 
what it was long ago, but then unjustly, called, a true science and 
art. No continuing success can be built on empiricism or upon the 
proportion of guesswork which is inseparable from dependence upon 
" the experienced eye." " Diagnosis " is now the password in med- 
ical science. A knowledge of electro-diagnosis, of ophthalmoscopy, 
of laryngoscopy, etc., is at the present day a sine qua non for accu- 
rate diagnosis. Equally important at all times, and frequently even 
more important, is a knowledge of clinical chemistry and micros- 
copy. It is inconceivable that a physician can rationally diagnos- 
ticate and treat diseases of the stomach, intestines, kidneys, and 
liver, etc., without laboratory facilities. 



Viil PREFACE TO THE FIRST EDITION. 

It has been the author's aim to present to students and physicians 
those facts in clinical chemistry and microscopy which are of practi- 
cal importance. With the hope of exciting interest in these unjustly 
neglected subjects, he has not confined himself to bare statements 
of facts, which must in themselves be dry and uninteresting, but he 
has attempted to point out the reasons which have led up to the 
conclusions reached. 

Chemical and microscopical methods are described in detail, so 
that the student and practitioner who has not had special training 
in such manipulations will be enabled to obtain satisfactory results. 

The subject-matter covers the examination of the blood, the secre- 
tions of the mouth, the gastric juice, feces, nasal secretion, sputum, 
urine, transudates, exudates, cystic contents, semen, vaginal dis- 
charges, and milk. In every case a description of normal material 
precedes the pathological considerations, which latter in turn are 
followed by an account of the methods used in examination. A 
glance at the table of contents will furnish an idea of the various 
subjects considered under each heading. 

In conclusion, it is the agreeable duty of the author to express 
his sincerest thanks to his wife for assistance without which this 
volume could not have been written, and likewise for those illustra- 
tions which are original ; to Dr. William H. Welch for his kind- 
ness in placing the former Hygienic Laboratory of the Johns 
Hopkins Hospital at his disposal during the years 1892 and 1893 ; 
to Dr. W. Milton Lewis for much valuable aid in the correction of 
the manuscript and proof-sheets ; and to Messrs. Lea Brothers & 
Co. for the typographical excellence of the work, the extremely 
satisfactory reproduction of the drawings, and for many acts of 
courtesy. 

CHARLES E. SIMON. 

Baltimore, Md., 1896. 



CONTENTS. 



CHAPTER I. 
THE BLOOD. 

PAGE 

General considerations 17 

General characteristics of the blood 17 

color 17 

odor . . . '.'.-..-.. 18 

specific gravity 18 

determination according to Roy . 18 

determination according to Hammerschlag 19 

determination according to Schmaltz and Peiper 19 

indirect estimation of the haemoglobin 20 

estimation of the solids of the blood 20 

reaction 21 

estimation of the alkalinity according to Landois-v. Jaksch ..... 22 

estimation of the alkalinity according to Lowy 24 

estimation of the alkalinity according to Engel 24 

estimation of the alkalinity according to Dare 25 

Chemical examination of the blood 27 

general chemistry of the blood 27 

coagulation of the blood 29 

blood-pigments 32 

hemoglobin and oxyhemoglobin 32 

haemoglobinaemia 36 

carbon monoxide haemoglobin 37 

nitric oxide haemoglobin 38 

hydrogen sulphide haemoglobin 38 

carbon dioxide haemoglobin . 38 

haematin 38 

haemin . 39 

methaemoglobin 40 

haematoidin 40 

haematoporphyrin 41 

the spectroscope 41 

the proteids of the blood 43 

the carbohydrates 44 

sugar 44 

estimation of the sugar in the blood 45 

"Williamson's diabetic blood test 46 

glycogen 46 

cellulose 47 

urea 47 

uraemia * 47 

ammonia 47 

uric acid and xanthin-bases - 48 

fat and fatty acids 50 

lactic acid • ? . 52 

ix 



X CONTENTS. 

Chemical examination of the blood — Continued. page 

biliary constituents 53 

acetone . . ,. 54 

Microscopical examination of the blood 54 

the red corpuscles . . . > 54 

variations in the size of the red corpuscles 54 

variations in the form of the red corpuscles , . . . 54 

variations in the color of the red corpuscles and color index .... 56 

variations in the number of the red corpuscles . . . 58 

behavior toward anilin dyes (polychromatophilia) 62 

granular degeneration 65 

Cabot's ring bodies ....... 67 

Ehrlich's hsemoglobinsemic Innenkorper 67 

nucleated red corpuscles 67 

the leucocytes 71 

general differentiation of the various forms of leucocytes 71 

differentiation of the leucocytes according to their behavior toward 

anilin dyes 73 

the small mononuclear leucocytes 73 

the large mononuclear leucocytes 75 

the polynuclear neutrophilic leucocytes 77 

the polynuclear eosinophilic leucocytes 79 

the mast cells 80 

the myelocytes ....... 81 

Ehrlich's pseud olymphocytes 83 

Turk's irritation forms 83 

iodophilia 83 

kucocytosis 85 

polynuclear neutrophilic hyperleucocytosis 86 

polynuclear neutrophilic hypoleucocytosis 96 

polynuclear eosinophilic hyperleucocytosis 100 

polynuclear eosinophilic hypoleucocytosis 104 

lymphocytosis 105 

lymphopenia 107 

variations in number of the large mononuclear leucocytes 107 

variations in number of the mast cells 108 

myelocytosis \ . 109 

the plaques 112 

the dust particles of Muller . 114 

General technique 114 

slides and cover-glasses 114 

the blood mount 115 

fixation 118 

The anilin dyes and principles of staining 119 

Methods of staining .... 123 

the eosinate of methylene-blue 123 

Ehrlich's triacid stain 124 

Pappenheim's triacid stains 126 

the Romanowsky methods . ' •• 127 

method of Nocht 127 



CONTENTS. xi 

Methods of staining — Continued. page 

method of Michaelis and Wolff 129 

method of Giemsa . 129 

method of Keuter 129 

method of Leishman 130 

method of Wright 130 

method of Koch 132 

method of Ewing 132 

method of Ziemann 133 

Ehrlich's eosin-methyiene-blue-methylal 133 

Michaelis' eosinmethylene-blue-acetone 134 

Strauss and Kohnstein's method 134 

staining with eosin. 135 

staining with Ehrlich's triglycerin mixture 135 

staining with Ehrlich's neutral mixture 135 

staining with Ehrlich's methyl-green-fuchsin 136 

staining wth Pappenheim's-methyl-green pyronin ' . . . 136 

staining with Ehrlich's dahlia ....:. 136 

Westphal's method 136 

the Chenzinsky-Plehn methods 137 

Futcher's carbol-thionin 137 

Ehrlich's hsematoxylin-eosin 138 

demonstration of iodophilia 138 

distribution of alkali 139 

Enumeration of the corpuscles of the blood 139 

method of Thoma-Zeiss - 139 

enumeration of the red cells 140 

enumeration of the leucocytes 143 

differential leucocyte counting 146 

enumeration of the plaques • • 147 

The hsematokrit 147 

Estimation of haemoglobin 150 

Dare's haemoglobinometer 151 

Fleischl's haemoglobinometer 153 

Oliver's hsemoglobinometer 155 

Gowers' hsemoglobinometer • • • : 157 

Talquist's method • 158 

Estimation of blood-iron . . . . ' • • 158 

Kryoscopic examination of the blood 163 

Bacteriology and parasitology of the blood 165 

typhoid fever = 165 

Widal's serum test • • • 165 

paratyphoid fever 170 

pneumonia * 171 

sepsis 173 

gonococcus septicaemia • • 176 

anthrax 177 

acute miliary tuberculosis - • 177 

glanders 178 

influenza • 178 



xii CONTENTS. 

Bacteriology and parasitology of the blood — Continued. page 

relapsing fever 179 

Malta fever 180 

yellow fever . 1 80 

bubonic plague 181 

malaria 182 

trypanosomiasis 191 

spotted fever (tick fever) 192 

filariasis 192 

distomiasis (bilharziasis) 195 

anguilluliasis 197 



CHAPTER II. 
THE SECEETIONS OF THE MOUTH. 

Saliva 198 

general characteristics 198 

chemistry of the saliva . 198 

microscopical examination of the saliva 200 

pathological alterations 202 

Special diseases of the mouth 203 

tuberculosis of the mouth 203 

actinomycosis 203 

catarrhal stomatitis 203 

ulcerative stomatitis 203 

gonorrheal stomatitis 203 

thrush 204 

Tartar 204 

Coating of the tongue 204 

Coating of the tonsils 205 

pharyngomycosis leptothrica 205 

tonsillitis 205 

glandular fever 206 

diphtheria 206 

scarlatina 209 

CHAPTER III. 
THE GASTEIC JUICE AND THE GASTRIC CONTENTS. 

PAGE 

The secretion of gastric juice 210 

Test-meals 211 

the test-breakfast of Ewald and Boas 211 

the test-dinner of Riegel 212 

the double test-meal of Salzer 212 

the test-breakfast of Boas • 212 

The stomach-tube 212 

contraindications to the use of the tube • 2 13 

introduction of the tube 213 



CONTENTS. xiii 

PAGE 

General characteristics of the gastric juice . 215 

amount 215 

Chemical examination of the gastric juice 216 

chemical composition of the gastric juice 216 

the acidity of the gastric juice 217 

determination of the acidity of the gastric juice 219 

source of the hydrochloric acid 221 

significance of free hydrochloric acid 222 

the amount of free hydrochloric acid 224 

euchlorhydria 224 

hypochlorhydria 224 

anachlorhydria 224 

hyperchlorhydria 225 

test for free acids 225 

test for free hydrochloric acid 226 

the dimethyl-amido-azo-benzol test 226 

the phloroglucin-vanillin test 226 

the resorcin test 227 

the tropseolin test 228 

Mohr's test 228 

the benzopurpurin test 228 

the combined hydrochloric acid 229 

quantitative estimation of the hydrochloric acid 230 

Topfer's method • . . 230 

Sahli's method 232 

Martius and Liittke's method . 232 

Leo's method 235 

the ferments of the gastric juice and their zymogens ^ . . 236 

pepsin and pepsinogen 236 

tests for pepsin and pepsinogen 238 

quantitative estimation 238 

chymosin and chymosinogen 239 

tests for chymosin and chymosinogen 240 

quantitative estimation 241 

the products of gastric digestion 241 

digestion of the native albumins 241 

digestion of the proteids 242 

digestion of the albuminoids 242 

digestion of the carbohydrates 242 

analysis of the products of albuminous digestion 244 

tests for the products of carbohydrate digestion 245 

lactic acid ." . 245 

mode of formation and clinical significance 245 

tests for lactic acid 248 

Uffelmann's test 248 

Killing's test 249 

Strauss' test 249 

Boas' test 250 

quantitative estimation of lactic acid according to Boas' method . . . 251 

the fatty acids r 253 



xiv CONTENTS. 

Chemical examination of the gastric juice — Continued. page 

mode of formation and clinical significance 253 

tests for butyric acid 254 

tests for acetic acid 255 

quantitative estimation of the fatty acids 255 

quantitative estimation of the organic acids 255 

gases 256 

acetone 258 

ptomains and toxalbumins . 258 

vomited material 259 

food-material 259 

mucus 260 

gastrosuccorrhoea mucosa 260 

saliva 261 

bile . . . • 261 

pancreatic juice . 261 

blood 261 

test of Miiller and Weber 261 

Donogany's method 262 

pus 262 

stercoraceous material 262 

parasites 263 

odor 263 

Microscopical examination of the gastric contents 264 

the Boas-Oppler bacillus 264 

sarcinse 264 

shreds of mucous membrane 265 

tumor particles 266 

Examination of the motor power of the stomach 266 

Leube's method 267 

the salol test of Ewald and Sievers 267 

Examination of the resorptive power of the stomach 268 

Indirect examination of the gastric juice 268 

Gunzburg's method 268 

Simon's method 269 

CHAPTER IV. 

THE FECES. 

PAGE 

Examination of normal feces 270 

general characteristics 270 

number of stools 270 

amount 270 

consistence and form 271 

odor 271 

color 271 

macroscopical constituents 272 

alimentary detritus 272 

foreign bodies 272 

microscopical constituents 272 



CONTENTS. xv 

Examination of normal feces — Continued. PAGE 

constituents derived from food 272 

morphological elements derived from the alimentary canal 274 

crystals 274 

parasites 275 

vegetable parasites 275 

fungi 276 

schizomycetes 276 

bacteria 276 

chemistry of normal feces 278 

reaction 278 



general composition 



_/ 



phenol, indol, and skatol . 279 

fatty acids -. 280 

cholesterin 282 

the biliary acids 283 

pigments 283 

xanthin bases , 285 

Pathology of the feces 285 

general characteristics 285 

number of stools 285 

consistence and form 285 

amount 286 

odor • .286 

reaction 286 

color 287 

macroscopical constituents . 289 

alimentary constituents 289 

mucus and mucous cylinders 290 

biliary and intestinal concretions 291 

analysis of gall-stones 292 

coproliths 292 

intestinal sand 292 

microscopical examination 293 

technique • 293 

remnants of food 293 

Schmidt's fermentation test 293 

Leiner's test for casein 295 

epithelium , 295 

red blood-corpuscles 296 

mucus 296 

leucocytes 296 

crystals 297 

animal parasites 297 

protozoa 298 

Amoeba coli 299 

Cercomonas hominis 301 

Trichomonas intestinalis 302 

Megastoma entericum 303 

Balantidium coli . 304 

vermes 305 

Taenia saginata 306 



xvi CONTENTS. 

Pathology of the feces — Continued. PAGE 

Taenia solium , 307 

Taenia nana 308 

Taenia diminuta 309 

Taenia cucumerina 309 

Taenia africana . . . „ 310 

Taenia madagascariensis 311 

Bothriocephalic latus 311 

Krabbea grandis 313 

Distoma hepaticum 313 

Distoma lanceolatum 314 

Distoma Buskii 314 

Distoma sibiricum 314 

Distoma spatulatum . . . 314 

Distoma conjunctum 315 

Distoma heterophyes 315 

Amphistomum hominis 315 

Ascaris lumbricoides 315 

Ascaris mystax 316 

Ascaris maritima 316 

Oxyuris vermicularis 316 

Anchylostomum duodenale 317 

Trichocephalus hominis 320 

Trichina spiralis 320 

Anguillula intestinalis 320 

insecta 323 

vegetable parasites 323 

bacillus of cholera 323 

Finkler-Prior bacillus ' 325 

typhoid bacillus 325 

tubercle bacillus 328 

Bacillus coli communis 328 

Bacillus lactis aerogenes 328 

Bacillus pyocyaneus 329 

Bacillus acidophilus 329 

Proteus vulgaris « 330 

Bacillus dysenteriae 331 

Chemistry of the feces 333 

ptoma'ins 334 

Meconium 334 



CHAPTER V. 

THE NASAL SECEETION. 

Physiology and pathology of the nasal secretion 336 



CONTENTS. xvii 

CHAPTER VI. 
THE SPUTUM. 

PAGE 

General technique 338 

General characteristics of the sputa 339 

amount „ 339- 

consistence . . 339 

color 340 

odor 340 

specific gravity 341 

configuration of sputa 341 

Macroscopical constituents of sputum 342 

elastic tissue 342 

fibrinous casts 342 

Curschmann's spirals 344 

echinococcus membranes 346 

concretions 346 

foreign bodies 346 

Microscopical examination 346 

leucocytes 346 

red blood-corpuscles . . 348 

epithelial cells 348 

elastic tissue 350 

animal parasites 352 

Taenia echinococcus 352 

Trichomonades 353 

Amoeba coli 353 

Distoma pulmonale . 353 

Distoma haematobium 354 

vegetable parasites 354 

pathogenic organisms 354 

the tubercle bacillus 354 

methods of staining 357 

Pappenheim's method 357 

Gabett's method 357 

Weigert-Ehrlich's method 358 

Ziehl-Neelsen's method 358 

cultivation of tubercle bacillus 359 

the Diplococcus pneumoniae 360 

the bacillus of influenza 360 

the bacillus of whooping-cough 361 

the smegma bacillus 361 

the typhoid bacillus 362 

the plague bacillus 362 

actinomycosis 362 

non-pathogenic organisms 363 

Oidium albicans ' 3(53 

Sarcina pulmonalis 363 

crystals 363 

Charcot-Leyden crystals 364 



xvm CONTENTS. 

Microscopical examination — Continued. PAGE 

hsematoidin ' 364 

cholesterin 365 

fatty acid crystals ; 365 

leucin and tyrosin 365 

calcium oxalate 365 

triple phosphates 365 

the pneumoconioses 365 

anthracosis . . 365 

siderosis 365 

chalicosis 366 

stycosis 366 

Chemistry of the sputum 366 



CHAPTEE VII. 
THE UEINE. 

General characteristics of the urine 369 

general appearance 369 

color 370 

odor 371 

consistence 371 

quantity 371 

polyuria 372 

oliguria 375 

specific gravity , 375 

determination of the specific gravity 378 

determination of the solid constituents 380 

Keaction 380 

determination of the acidity of the urine 384 

Freund's method 384 

Chemistry of the urine 385 

general chemical composition of the urine 385 

quantitative estimation of the mineral ash of the urine 386 

the chlorides 387 

test for chlorides in the urine 390 

quantitative estimation of the chlorides by the method of Salkowski- 

Volhard 390 

direct method 394 

estimation of the chlorides after incineration (according to Neubauer 

and Salkowski) • 395 

the phosphates 395 

test for the phosphates in the urine 400 

quantitative estimation of the total amount of phosphates 401 

separate estimation of the earthy and alkaline phosphates ...... 404 

removal of the phosphates from the urine 404 

the sulphates 405 

test for the sulphates in the urine 408 

quantitative estimation of the sulphates 409 



CONTENTS. xix 

Chemistry of the urine — Continued. page 

quantitative estimation of the total sulphates 409 

quantitative estimation of the conjugate sulphates 410 

neutral sulphur 411 

quantitative estimation . 413 

urea 414 

properties of urea 422 

urea nitrate 423 

urea oxalate 424 

separation of urea from the urine 425 

quantitative estimation of urea 426 

estimation of nitrogen according to Kjeldahl 435 

estimation of nitrogen according to Will-Varrentrapp 437 

ammonia 439 

quantitative estimation 441 

Schlosing's method 441 

Folin's method 441 

uric acid 442 

properties of uric acid 448 

tests for uric acid 449 

quantitative estimation of uric acid 449 

xanthin-bases 455 

quantitative estimation 456 

hippuric acid 457 

properties of hippuric acid 458 

quantitative estimation of hippuric acid 459 

kreatin and kreatinin 460 

properties of kreatin and kreatinin 461 

test for kreatinin in the urine . 462 

quantitative estimation of kreatinin in the urine 462 

oxalic acid 466 

properties of oxalic acid 469 

tests for oxalic acid 469 

quantitative estimation of oxalic acid 470 

Albumins 472 

serum-albumin 472 

Patein's or aceto-soluble albumin 485 

serum-globulin 485 

albumoses (peptones) 486 

Bence Jones' albumin 487 

peptone 489 

haemoglobin 489 

fibrin 490 

nucleo-albumin 491 

- histon and nucleohiston 492 

tests for albumin 492 

tests for serum-albumin 492 

nitric acid test 493 

boiling test 496 

potassium ferrocyanide test 497 

trichloracetic acid test 497 



xx CONTENTS. 

Albumins — Continued. PAGE 

picric acid test 498 

Spiegler's test 498 

special test for serum-albumin 498 

quantitative estimation of albumin 499 

old method of boiling 499 

volumetric method of Wassiliew 499 

Esbach's method 500 

differential density method 500 

gravimetric method 501 

test for serum-globulin and its quantitative estimation 502 

tests for albumoses \ 502 

Salkowski's method 502 

Bang's method 503 

examination for peptone 504 

tests for Bence Jones' albumin 504 

tests for (mucin) nucl^o-albumin 505 

tests for haemoglobin * 506 

Heller's test 507 

the guaiacum test . 507 

Donogany's test 507 

test for fibrin 508 

test for histon 508 

Carbohydrates . . . . ' 508 

glucose 508 

tests for sugar 517 

Trommer's test 517 

Fehling's test 518 

Bottger's test with Nylander's modification 518 

fermentation test 519 

phenylhydrazin test 520 

polarimetric test 521 

quantitative estimation of sugar 522 

Fehling's method 523 

Knapp's method 525 

differential density method 526 

Einhorn's method 526 

Lohnstein's method 527 

polarimetric method 528 

Bremer's diabetic urine test 530 

lactose • 531 

levulose 531 

maltose 531 

dextrin 532 

laiose - 532 

pentoses 532 

Tollens' orcin test 532 

Tollens' phloroglucin test 533 

animal gum 533 

Glucuronic acid 533 



CONTENTS. xxi 

PAGE 

Inosit 534 

Urinary pigments and chromogens 534 

normal pigments 535 

urochrome 535 

uroerythrin 536 

normal chromogens 537 

indican , 537 

tests for indican 540 

quantitative estimation 541 

urohaematin 543 

urorosei'nogen 544 

pathological pigments and chromogens 545 

blood-pigments 545- 

hsematin 545 

urorubrohsematin and urofuscohaematin 545 

urohsematoporphyrin 545 

biliary pigments 548 

Smith's test 550 

Huppert's test 550 

Gmelin's test (as modified by Kosenbach ) 550 

Gmelin's test 550 

biliary acids 550 

cholesterin 551 

pathological urobilin 551 

melanin and melanogen 554 

phenol urines 555 

alkapton 555 

homogentisinic acid . . . 555 

blue urines . - 559 

green urines . . 559 

pigments referable to drugs 559 

Ehrlich's diazo-reaction 559 

benzaldehyde reaction 564 

Conjugate sulphates 566 

skatoxyl 566 

phenol 566 

Salkowski's test 566 

quantitative estimation 567 

pyrocatechin 567 

Acetone 568 

tests for acetone 569 

Legal's test 569 

Lieben's test 570 

Keynolds' test 570 

Stock's test 570 

Dennige's test 570 

quantitative estimation 571 

Diacetic acid 573 

Arnold's test 573 

Oxybutyric acid . '. 574 

Crotonic acid . 576 



xxii CONTENTS. 

PAGE 

Lactic acid 576 

Oxyamygdalic acid 577 

Volatile fatty acids - 577 

Fats „ ; 578 

chyluria 579 

galacturia , 579 

Ferments 579 

Gases 580 

hydrothiormria 580 

Ptomai'ns 581 

method of examination for ptomai'ns . 583 

Kryoscopic examination of the urine 585 

Sediments 585 

Microscopical examination of the urine 585 

non-organized sediments 588 

sediments occurring in acid urines 588 

uric acid 588 

amorphous urates •■• 589 

calcium oxalate 590 

ammonio-magnesium phosphate 591 

monocalcium phosphate 592 

neutral calcium phosphate 593 

basic magnesium phosphate 593 

hippuric acid 593 

calcium sulphate 593 

cystin 594 

leucin and tyrosin 595 

xanthin ........... 598 

soaps of lime and magnesia 598 

bilirubin and hsematoid in 599 

fat 599 

sediments occurring in alkaline urines 600 

basic phosphate of calcium and magnesium 600 

ammonium urate • . • 601 

magnesium phosphate 601 

ammonio-magnesium phosphate . . . . 601 

calcium carbonate 601 

indigo 601 

organized constituents of urinary sediments 602 

epithelial cells 602 

leucocytes 605 

red blood-corpuscles 610 

tube-casts 612 

true casts 614 

hyaline casts 614 

waxy casts 616 

pseudo-casts * 618 

cylindroids 618 

formation of tube-casts 618 

clinical significance of tube-casts 619 



CONTENTS. xxiii 

Microscopical examination of the urine — Continued. page 

spermatozoa ....... . 622 

parasites 623 

vegetable parasites , 623 

animal parasites . <? « 628 

tumor particles ...«,. 630 

foreign bodies 630 

CHAPTER VIII. 

TRANSUDATES AND EXUDATES. 

Transudates 631 

general characteristics 631 

specific gravity < 631 

chemistry of transudates 633 

microscopical examination 634 

Exudates 634 

serous exudates 634 

hemorrhagic exudates » 634 

tuberculosis 635 

cancer - 636 

technique 637 

bacteriological examination of 637 

chemistry of - 638 

pus , , . 640 

general characteristics of pus .................. 640 

chemistry of pus 640 

microscopical examination of pus 641 

leucocytes 641 

giant corpuscles 642 

detritus 642 

red blood-corpuscles 642 

pathogenic vegetable parasites ................ 642 

v protozoa ................. • 643 

vermes 643 

crystals. ........."..'.........■...-•,"• 643 

technique 643 

gonorrhceal pus 644 

the gonococcus ..... ......... 644 

putrid exudates 647 

chylous and chyloid exudates 647 



CHAPTER IX. 

THE CEKEBEOSPINAL FLUID. 

Amount 650 

Appearance 650 

Specific gravity 651 



xxiv CONTENTS. 

PAGE 

Reaction 652 

Chemical composition . . ■ 652 

Microscopical examination 653 

Bacteriology .......-.'... . ...... . 654 

Toxicity . . . 656 

CHAPTER X. 

THE EXAMINATION OF CYSTIC CONTENTS. 

Cysts of the ovaries and their appendages 657 

Hydatid cysts 659 

Hydronephrosis 659 

Pancreatic cysts 660 

CHAPTER XI. 

THE SEMEN. 

General characteristics , 661 

Chemistry of the semen 661 

Microscopical examination of the semen 662 

Pathology of the semen 663 

The recognition of semen in stains 664 

CHAPTER XII. 
VAGINAL DISCHARGES. 

Bacteriology 666 

Vaginal blennorrhea 668 

Menstruation 668 

The lochia 668 

Vulvitis and vaginitis 669 

Membranous dysmenorrhea 669 

Cancer 669 

Gonorrhoea 669 

Abortion 670 

CHAPTER XIII. 

THE SECRETION OF THE MAMMARY GLANDS. 

The secretion of milk in the newly born 672 

Colostrum , 672 

The secretion of milk in the adult female 673 

Human milk . . . . 673 

The milk in disease 674 

determination of the specific gravity 675 

estimation of the fat 677 

estimation of the proteids 677 



CLINICAL DIAGNOSIS. 



CHAPTER I. 
THE BLOOD. 

GENERAL CONSIDERATIONS. 

If blood is allowed to flow directly from an artery into a vessel 
surrounded by a freezing-mixture, and containing one-seventh its 
volume of a saturated solution of sodium sulphate, or a 25 per 
cent, solution of magnesium sulphate (1 volume to 4 volumes 
of blood), it will be observed that after some time a sediment, 
presenting the color of arterial blood, has formed at the bottom, 
which is covered by a layer of clear, straw-colored fluid- — the blood- 
plasma. Upon microscopical examination the sediment will be seen 
to contain : 

a. Numerous homogeneous, non-nucleated, circular, biconcave 
disks. These measure on an average 7.5 y. in diameter, and are of a 
faint greenish-yellow color when viewed through the microscope, 
while en masse they present the color of arterial blood — the erythro- 
cytes or red corpuscles of the blood. 

b. Roundish or irregularly shaped nucleated cells which are for 
the most part granular and far less numerous than the red corpus- 
cles, and devoid of coloring-matter — the leucocytes, colorless or 
white corpuscles of the blood. 

c. Minute colorless disks, measuring less than one-half the di- 
ameter of a red corpuscle — the so-called blood-plaques, or blood- 
plates of Bizzozero. 

GENERAL CHARACTERISTICS OF THE BLOOD. 

Color. 

Chemical examination of the blood shows that its color is ref- 
erable to the presence of an albuminous, iron-containing substance — 
haemoglobin — in the bodies of the red corpuscles, which is characterized 
by its great avidity for oxygen, and forms a compound therewith, 
known as oxyhemoglobin. The relatively larger amount of the 
2 17 



18 THE BLOOD. 

latter encountered in the arteries, as compared with the veins, causes 
the difference in the appearance of arterial and venous blood, the 
former presenting a bright scarlet-red, the latter a dark-bluish color. 
A bright cherry-red color is noted in cases of poisoning with carbon 
monoxide, while a brownish-red or chocolate color is observed in 
cases of poisoning with potassium chlorate, anilin, hydrocyanic 
acid, and nitrobenzol. A milky appearance is frequently seen in 
cases of well-marked leukaemia. In chlorosis and hydraemic con- 
ditions, as would be expected, the blood is pale and watery. 

Odor. 

The peculiar odor of the blood, which varies in different animals, 
the halitus sanguinis of the ancients, is due to the presence of 
certain volatile fatty acids, and may be rendered more distinct by 
the addition of concentrated sulphuric acid. 

Specific Gravity. 

The specific gravity of the blood in healthy adults varies between 
1.058 and 1.062, being higher on an average in men, 1.059, than 
in women, 1.05(3, and children — boys 1.052, girls 1.050. Gen- 
erally speaking, it is proportionate to the amount of haemoglobin and 
the volume of the red corpuscles. It is diminished to a certain 
extent by fasting, the ingestion of solids and liquids, gentle exercise, 
pregnancy, etc. The specific gravity, moreover, depends upon the 
bloodvessel from which the specimen is taken, being higher, gen- 
erally speaking, in venous than in arterial blood. 

Under pathological conditions the specific gravity may vary 
between 1.025 and 1.068. In nephritis, chlorosis, the anaemias in 
general, and in cachectic conditions (carcinoma of the stomach, etc.) 
it may diminish to 1.031. In pulmonary phthisis the specific 
gravity is diminished in the third stage (1.040-1.042), and in the 
first stage (1.049) in the case of those patients in whom the onset 
has been very gradual. In the second stage normal figures are 
obtained (1.058-1.060), corresponding to the relatively high per- 
centage of haemoglobin (90-95 per cent.) which is then noted, and 
which is referable no doubt to an actual concentration of the blood 
(Appelbaum). An increased specific gravity is met with in febrile 
diseases (typhoid fever, 1.057 to 1.063), conditions associated with 
pronounced cyanosis (emphysema, fatty heart, uncompensated val- 
vular disease, 1.054 to 1.068), and obstructive jaundice, 1.062. 

Methods of Determining the Specific Gravity of the Blood. 
— Roy's Method. — A number of test-tubes are filled with a mixt- 
ure of glycerin and water in different proportions, so that the specific 
gravity in the different tubes varies between 1.025 and 1.068. 



GENERAL CHARACTERISTICS OF THE BLOOD. 19 

Blood is then drawn from the tip of a finger, or the lobe of the ear, 
into a capillary tube connected with an ordinary hypodermic syringe, 
pressure being avoided. A drop of blood is placed in each tube, in 
which it will sink as long as the specific gravity of the glycerin 
mixture is lower than that of the blood, while it will remain sus- 
pended in a mixture the specific gravity of which is equivalent 
to its own. 

Roy states that it is important for the purpose of comparison to 
make such examinations in each case at the same hour, as the spe- 
cific gravity of the blood has been shown to undergo diurnal varia- 
tions. 

Hammerschlag's Method. — A cylinder, measuring about 10 cm. 
in height, is partly filled with a mixture of chloroform (sp. gr. 1.526) 
and benzol (sp. gr. 0.889), having a specific gravity of 1.050 to 
1.060. Into this solution a drop of blood is allowed to fall 
directly from the finger, pressure being avoided, and care taken 
that the drop does not come in contact with the walls of the vessel. 
The drop, moreover, should not be too large, as otherwise it will 
separate into droplets, giving rise to inaccurate results. Should 
the drop sink to the bottom, it is apparent that the specific gravity 
of the mixture is lower than that of the blood, necessitating 
the addition of chloroform. This should be added drop by drop 
while the mixture is thoroughly stirred. If, on the other hand, the 
drop should tend toward the surface, it is best to add an amount of 
benzol sufficient to cause the blood to sink to the bottom, and then to 
bring it to the proper degree of suspension by the subsequent addi- 
tion of chloroform. As soon as the drop remains suspended the 
mixture is filtered, and its specific gravity ascertained by means of 
an accurate areometer registered to the fourth decimal. The figure 
obtained is the specific gravity of the blood. The chloroform- 
benzol mixture may be kept indefinitely. 

With practice, results sufficiently accurate for clinical purposes 
may thus be obtained with an expenditure of very little time. 

Instead of the chloroform-benzol mixture, one of chloroform and 
olive oil may be employed, as suggested by van Spanje. It has 
the advantage of being less volatile than the other. Three parts of 
chloroform and one of the oil give a mixture with a specific gravity 
of 1.056. 

Schmaltz and Peiper's Method. — Where delicate scales are avail- 
able the method of Schmaltz and Peiper may be employed, and is 
certainly the most accurate : a capillary tube, measuring about 12 
cm. in length and 1.5 mm. in width, with its ends tapering to a 
diameter of 0.75 mm., is filled with blood and carefully weighed, 
when the weight of the blood, divided by the weight of an equiva- 
lent volume of distilled water, will indicate the specific gravity. 

As the result of numerous investigations it may now be regarded 



20 THE BLOOD. 

as an established fact that with the exception of nephritis, circulatory 
disturbances, leukaemia, and possibly also post-hemorrhagic anaemia 
and that resulting from inanition, the specific gravity of the blood 
varies directly with the amount of haemoglobin and the volume of 
the red corpuscles. A simple method is thus given by means of 
which haemoglobin estimations can usually be made in the absence 
of more expensive instruments. In the following table the specific 
gravities, as obtained with Hammerschlag' s method, and that of 
Schmaltz and Peiper, are given, with the corresponding amounts of 
haemoglobin ; the figures, however, are probably not quite accurate : 

Specific gravity Specific gravity 

according to Haemoglobin. according to Haemoglobin. 

Hammerschlag. Schmaltz and Peiper. 

1.033-1.035 . . . 25-30 per cent, 1.030 20 per cent. 

1.035-1.038 . . . 30-35 " 1.035 30 " 

1.038-1.040 . . . 35-40 " 1.038 35 " 

1.040-1.045 . . . 40-45 " 1.041 40 " 

1.045-1.048 . . . 45-55 " 1.0425 45 " 

1.048-1.050 . . . 55-65 " 1.0455 50 " 

1.050-1.053 . . . 65-70 " 1.048 55 " 

1.053-1.055 . . . 70-75 " 1.049 60 " 

1.055-1.057 . . . 75-85 " 1.051 65 " 

1.057-1.060 . . . 85-95 " 1.052 70 " 

1.0535 75 " 

1.056 80 " 

1.0575 90 " 

1.059 100 " 

Literature. — Schmaltz, Deutsch. Arch. f. klin. Med., vol. xlvii. p. 145; and 
Deutsch. med. Woch., 1891, No. 16. Stintzing u. Gumprecht, Deutsch. Arch. f. klin. 
jvled., vol. liii. p. 265. Siegl, Prag. med. Woch., 1892, No. 20 ; and Wlen. med. Woch., 
1891, No. 33. Hammerschlag, Ibid., 1890, p. 1018; and Zeit. f. klin. Med., 1892, vol. 
xxii. p. 475. Schmaltz, Deutsch. Arch. f. klin. Med., 1890, vol. xlvii. p. 145 ; and Deutsch. 
med. Woch.. 1891, vol. xvii. p. 555. Appelbaum, Berl. klin. Woch., 1901, vol. xxxix. p. 7. 

Direct Estimation of the Solids of the Blood. — A few drops 
of blood (0.2 to 0.3 gramme), obtained by means of a fairly deep 
incision or puncture into the tip of a finger, moderate pressure being 
made upon the middle phalanx if necessary, are collected in a watch- 
crystal. This is at once covered with its fellow and weighed. The 
specimen (open) is then dried at a temperature of from 60° to 70° C. 
for twenty-four hours, and again weighed, the weight of the solids 
being thus ascertained. 

In healthy adults the following values were obtained by Stintzing 
and Gumprecht : 

Average. 

In men 21.6 

In women 19.8 

In conditions associated with chronic anaemia the solids, as would 
be expected, are always much diminished. In leukaemia, on the 
other hand, owing to the large number of leucocytes present, a rela- 
tive increase is observed. 



Maximum. 


Minimum. 


Average water. 


23.1 


19.6 


78.4 per cent. 


21.5 


18.4 


80.2 



GENERAL CHARACTERISTICS OF THE BLOOD. 21 

Reaction. 

The reaction of the blood during life, owing to the presence of 
disodium phosphate and sodium carbonate, is alkaline, the degree of 
alkalinity in terms of sodium hydrate under normal conditions cor- 
responding to 182 to 218 mgrms. for every 100 c.c. of blood, v. 
Jaksch gives 260 to 300 mgrms. as the normal, and Canard 203 to 
276 mgrms. 

The alkaline reaction of the blood may be demonstrated by re- 
peatedly drawing a strip of red litmus-paper, thoroughly moistened 
with a concentrated solution of common salt, through the blood, 
and rapidly washing off the corpuscles with the same solution, when, 
as a general rule, the alkaline reaction can be clearly made out. 

Small plates of plaster of Paris or clay, stained wdth neutral 
litmus solution, may be similarly employed, the blood in this case 
being washed off with water. 

Generally, the alkalinity of the blood is lower in women and 
children than in men, and is, furthermore, influenced by the proc- 
ess of digestion, exercise, etc. At the beginning of digestion, 
when hydrochloric acid is being freely secreted, the alkalinity of 
the blood increases ; while later on, when both hydrochloric acid 
and peptones are reabsorbed, the alkalinity in turn diminishes. 
Higher values are usually found during pregnancy than in the non- 
pregnant state. 

A decrease is observed following violent muscular exercise, such 
as forced marches by soldiers, owing, in all probability, to an exces- 
sive production of acids in the muscles. 

Under pathological conditions a diminished alkalinity of the blood 
is frequently observed. This is particularly marked in cases of 
severe anaemia (108 to 145 mgrms. of NaOH), and increases as the 
number of red corpuscles and the amount of haemoglobin diminish. 
In chlorosis, however, the diminution in the number of red corpus- 
cles is accompanied by a normal, or but slightly diminished, alkalinity 
of the blood as a whole. In leukaemia, pernicious anaemia, nephritis 
when accompanied by uraemia, various hepatic affections, diabetes, 
carcinoma, the various profound cachexia?, pseudoleukaemia, poison- 
ing with carbon monoxide and acids, and finally in highly febrile 
conditions, as in typhoid fever, and toxic processes in general, the 
alkalinity of the blood is diminished, the lowest value found corre- 
sponding to 108 mgrms. of KaOH. A similar decrease follows the 
prolonged use of acids, while an increase is brought about by the 
ingestion of alkalies. An increase in the alkalinity of the blood 
occurs after a cold bath, and it is interesting to note that this is 
apparently associated with an increase in the bactericidal power of 
the blood. Possibly the beneficial effect of cold baths in fever 
may be explained upon this basis. The supposition that in gout 



22 THE BLOOD. 

a diminished alkalinity exists in the intervals between the attacks, 
and that this increases beyond the normal during the attack, has 
been proved unfounded. 

v. Jaksch employs the following method, a modification of that 
originally devised by Landois : eighteen watch-crystals are prepared, 
each containing a mixture of a concentrated solution of sodium 
sulphate and a y^ and a yoVo normal solution of tartaric acid, in 
varying proportions, so that crystal 

No. C.c. Cc. 

I. Shall contain 0.9 of the T ^o norm. sol. of the acid, and 0.1 of the cone. Na 2 S0 4 sol. 



II. 


a 


" 


0.8 


" 


" 


a 


" 


a 


" 


0.2 


III. 


" 


a 


0.7 


(i 


" 


a 


" 


a 


it 


0.3 


IV. 


" 


a 


0.6 


a 


a 


it 


it 


a 


" 


0.4 


V. 


it 


" 


0.5 


a 


it 


it 


a 


a 


it 


0.5 


VI. 


a 


it 


0.4 


it 


it 


a 


a 


tt 


a 


0.6 


VII. 


it 


" 


0.3 


a 


a 


a 


a 


a 


it 


0.7 


VIII. 


a 


a 


0.2 


tt 


a 


a 


« 


tt 


a 


0.8 


IX. 


a 


a 


0.1 


a 


a 


it 


a . 


tt 


it 


0.9 


X. 


tt 


a 


0.9 


a 


Too"o 


tt 


tt 


tt 


tt 


0.1 


XI. 


tt 


tt 


0.8 


a 




tt 


tt 


tt 


tt 


0.2 




etc., 


for each c.c. 


of the mixture. 











Blood is taken, preferably from the back, by means of cupping- 
glasses, and, before it coagulates, 0.1 c.c. is added to each c.c. of 
the mixture described, when the reaction is determined in each 
crystal by means of very sensitive litmus-paper. The amount of 
acid contained in the specimen exhibiting a neutral reaction in terms 
of NaOH will then indicate the degree of alkalinity of the blood. 

As 150 (molecular weight) parts by weight of tartaric acid (C 4 H 6 6 ) 
combine with 80 (molecular weight) parts by weight of JNaOH, or 
75 with 40, according to the equation : 

/COOH /COONa 

C 2 H 2 (OH) 2 < + 2NaOH = C 2 H 2 (OH) 2 < + 2H 2 0, 

\COOH XX)ONa 

a normal solution would contain 75 grammes of pure tartaric acid 
to the liter and a y^-g- and a ywu~& norma l solution, respectively, 0.75 
and 0.075 gramme. As 1000 c.c. of a y-^ normal solution would 
correspond to 0.4 gramme of NaOH, and 1000 c.c. of a yoVo^ nor " 
mal solution to 0.04 gramme, 1 c.c. of the t |q- normal solution will 
represent 0.0004, and 1 c.c. of the y^oo" norma l solution 0.00004 
gramme of NaOH. 

Supposing, then, that a neutral reaction was obtained in the crystal 
containing 0.6 c.c. of the y^- normal solution, the alkalinity of the 
0.1 c.c. of blood in terms of NaOH would correspond to 0.00024 
gramme of NaOH, or 0.24 gramme for 100 c.c. of blood. 

As the alkalinity of the blood rapidly diminishes after being 
drawn, owing, in all probability, to the formation of an acid caused 



GENERAL CHARACTERISTICS OF THE BLOOD. 23 

bv decomposition of the haemoglobin, it is apparent that the ex- 
periment must be performed as rapidly as possible, and not more 
than one minute and a half should elapse between the withdrawal 
of the blood .and the conclusion of the experiment. 

Until comparatively recently this method was the only one avail- 
able for clinical purposes, and the results detailed above were 
obtained by its aid. It is open to numerous objections, however, 
and is too complicated for routine work. Of late, a method 
suggested by Lowy has attracted much attention, and, to judge 
from the literature, is destined soon to replace the one described. 
It is both simpler and more accurate. The results, however, differ 
considerably from those given above, and a careful revision of the 
work thus far accomplished with the old method will be necessary 
before definite conclusions can be reached. In healthy adults while 
fasting the alkalinity of the blood, according to Lowy, corresponds 
to about 300 to 325 mgrms. of sodium hydrate for every 100 c.c. 
of blood. Variations amounting to 75 mgrms., plus or minus, are, 
however, not uncommon, and, according to Strauss, the unavoidable 
errors may correspond to 30 mgrms. Some of the results obtained 
in disease are here given : 

Carcinoma cesophagi 227-643 

Carcinoma ventriculi 256-635 

Ulcus ventriculi 302-460 

Anadeny of the stomach 354-360 

Alcoholic gastritis ' 343-379 

Chronic enteritis 212-272 

Phthisis pulmonalis 450-468 

Bronchitis 239-343 

Neurasthenia 225-426 

Arteriosclerosis 208-344 

Chronic arthritis 368-465 

Erysipelas 498 

Typhoid fever 270-640 

Pneumonia 263-464 

Septicaemia 443 

Leukaemia 368-835 

Pernicious anaemia 429 

Diabetes mellitus 362-457 

Chronic interstitial nephritis 310-409 

Chronic parenchymatous nephritis 312-490 

Cirrhosis of the liver 272-345 

A constant diminution of the alkalinity of the blood was noted by 
Brandenburg in anemic conditions (202-239 mgrms. of NaOH), 
while the total amount of the albumins was at the same time 
diminished. An increase of both factors occurred in catarrhal jaun- 
dice ; variable results were obtained in two cases of typhoid fever 
and in one of pyaemia. In uraemia a material decrease was observed 
which was not associated with a decrease of the total albumins. 

According to Orlowsky, the variations in the alkalinity of the 
blood which have been noted in various diseases and sometimes in 



24 THE BLOOD. 

one and the same disease, by various investigators working with the 
older methods, are referable to the varying isotonicity of the blood 
and its varying richness in red corpuscles. Working with blood- 
plasma Orlowsky found a marked diminution of the alkalinity in 
advanced uraemia, in cancerous cachexia, and in severe cases of 
diabetes, while in other diseases normal values or at most but slight 
and exceptional variations were observed. 

Lowy's Method. — Five c.c. of blood, obtained from one of the 
superficial veins of the arm (preferably the median cephalic), are 
allowed to flow into a small flask provided with a long and partially 
graduated neck, and containing 45 c.c. of a 0.25 per cent, solution 
of ammonium oxalate. Coagulation is thus prevented and the blood 
made lake-colored — i. e., the haemoglobin is dissolved from the 
stroma of the red corpuscles. The mixture is then titrated with a 
-J5- normal solution of tartaric acid, using lacmoid paper, soaked in a 
concentrated solution of magnesium sulphate, as an indicator. The 
lacmoid paper is prepared as follows : 

A mixture of 100 grammes of resorcin, 5 grammes of sodium 
nitrite, and 5 c.c. of distilled water, is heated on an oil-bath to a 
temperature of 110° C. A violent reaction occurs at this point, and 
the flame should be removed before it is reached. The substance 
is then heated to a temperature of 115°-120° C. until all the am- 
monia which is evolved during the process has been driven oif. The 
residue, which should be of a pure blue color, is dissolved in water 
and precipitated with hydrochloric acid. On cooling, the coloring- 
matter is filtered off with the aid of a suction-pump, and washed 
with a little water. It is then dissolved in absolute alcohol, filtered, 
and the solution allowed to evaporate in an atmosphere free from 
ammonia. One gramme of the pigment, which crystallizes in 
reddish-brown, glistening platelets, is dissolved in 1000 c.c. of 45 
per cent, alcohol ; in this solution strips of fine Swedish filter-paper 
are soaked and then allowed to dry. 

As a normal solution of tartaric acid contains 75 grammes to the 
liter (see page 22), a -^ normal solution will contain 3 grammes, 
and 1 c.c. of the^ -^ normal solution will correspond to 0.0016 
gramme of sodium hydrate. 

Supposing, then, that 10 c.c. of the -^ normal solution were 
necessary to neutralize the 5 c.c. of blood, the alkalinity of these 5 
c.c. in terms of sodium hydrate would correspond to 0.016 gramme, 
and the alkalinity of 100 c.c. of blood to 0.016 X 20 = 0.320 
gramme — i. e., to 320 mgrms. 

Engel's Method. — This is essentially a modification of Lowy's 
method, and is well adapted for clinical purposes, as the amount of 
blood which is required for a single examination can readily be 
obtained by ordinary puncture. 



GENERAL CHARACTERISTICS OF THE BLOOD. 



25 



The blood is measured and rendered lake-colored in a specially 
constructed pipette (Fig. 1). To this end, the blood is drawn to 
the 0.05 c.c. mark and diluted with neutral distilled water, so that 
the volume of the mixture reaches the 5 c.c. line. After slight 



Fig. 1. 




Engel's alkalimeter. 

agitation the solution is placed in a small beaker and is titrated 
with a -^ normal solution of tartaric acid, from a special burette 
which accompanies the pipette. This is so constructed that each 
cubic centimeter is divided into twenty parts. Before and after the 
addition of every drop of the titrating fluid the reaction of the 
mixture is tested by placing a drop upon Lowy's lacmoid paper (see 
above). The end-reaction is reached when the yellow drop of the 
blood mixture shows a distinct red line along the margin. The result 
is expressed in terms of milligrammes of sodium hydrate per 1 c.c. 
of blood. Normally about 10 c.c. of the acid solution are em- 
ployed. The tartaric acid solution contains 1 gramme to the liter, 
so that 1 c.c. corresponds to 0.533 mgrm. of sodium hydrate. 

Supposing that 0.6 c.c. of the acid solution was required to neu- 
tralize the 0.05 c.c. of blood, then 12 c.c. would be necessary for 
1 c.c. of blood. As 1 c.c. of the acid solution represents 0.533 
mgrm. of sodium hydrate, the alkalinity of 1 c.c. of blood would 
correspond to 12 X 0.533 — i.e., to 6.396 mgrms. 

Dare's Method. — This method is based upon the fact that the 
characteristic spectrum of oxyhemoglobin disappears at the point 
of exact neutralization when the blood is titrated with a dilute solu- 
tion of tartaric acid. 



26 



THE BLOOD. 




The examination is made with the aid of a special instrument, the 

hcemoalkalimeter, which is pictured in the accompanying illustration 

(Fig. 2). B is a glass stopper through which passes an automatic 

capillary blood pipette of 20 cbmm. capacity, the exposed end 

of which is ground to a tapering point. The 

Fig. 2. stopper fits into the tube A, which has a capacity 

/'.:::::, of 3 c.c. and is graduated in cubic centimetres. 

\j The upper end of the tube is blown into a bulb 

ll « with a minute aperture at C. A 2 c.c. dropping- 
tube provided with a short piece of rubber tubing 
accompanies the instrument. 

To neutralize the blood, a -^-J-q- normal solu- 
tion of tartaric acid is used, which should con- 
tain an amount of alcohol sufficient to prevent 
the growth of bacteria, but insufficient to pre- 
cipitate the albumins of the blood. The reagent 
may be prepared by dissolving 0.075 gramme 
of tartaric acid (Merck's crystals ; guaranteed 
reagent) in a small amount of distilled water, 
adding 20 c.c. of alcohol (93—94 per cent.), and 
diluting to 200 c.c. with water. 

For the spectroscopic examination a Browning 
instrument (Fig. 12) will suffice. 

Method. — -A drop of blood is obtained from 
the finger-tip or the lobe of the ear in the usual 
manner. The blood pipette is filled in situ by 
capillary attraction, holding the instrument hori- 
zontally to the drop of blood as it emerges- from 
the wound. With an ordinary medicine-dropper filled with dis- 
tilled water the blood is washed into the bottom of the tube, con- 
necting the dropper with the pipette by means of a short piece of 
rubber tubing. Blood and water should just reach the zero mark, 
and are intimately mixed by closing the aperture in the bulb with 
the finger and inverting the tube several times. The reagent pipette 
is then filled with the tartaric acid solution and the rubber tubing 
slipped over the outer end of the blood pipette ; by compressing the 
rubber bulb the acid solution is forced through the pipette into the 
test-tube, the aperture in the glass bulb being closed before the 
pressure is relaxed. Having done this the tube is inverted several 
times while still attached to the reagent pipette, taking care that 
this is held vertically, so that the acid solution does not get into 
the rubber bulb. The tube is clamped in front of the spectroscope 
and examined for the two bands of oxyhemoglobin (Fig. 4). So 
long as these are visible more of the acid is added, inverting the 
tube after each addition ; as the bands become fainter one drop at 
a time is allowed to enter. At first this is rather tedious, but after 



Dare's hsemoalkalimeter. 



CHEMICAL EXAMINATION OF THE BLOOD. 27 

several examinations have been made it will be found unnecessary 
to apply the spectroscope so frequently to determine the point of 
neutralization, as the eye rapidly learns to recognize this by the 
characteristic change of color of the blood mixture. The observation 
is at an end when the oxyhemoglobin bands have just disappeared. 

The examination is made with artificial light, keeping the distance 
from the light constant. 

Dare suggests that for sake of convenience the results be expressed 
in terms of the number of cubic centimetres of the tartaric acid 
solution instead of in mgrms. of sodium hydrate, as has been cus- 
tomary. The corresponding values are given in the table below, 
and have reference to 100 c.c. of blood. His normal values range 
between 266 and 292. 

Equivalent in terms 
C.c. of reagent: of mgrms. of KaOH 

per 100 c.c. of blood. 

2.6 345.0 

2.4 319.0 

2.2 .... 292.0 

2.0 ■ . . 266.0 

1.8 239.0 

1.6 212.0 

1.4 176.0 

1.2 169.0 

1.0 133.0 

0.8 96.0 

0.6 79.0 

0.4 53.0 

0.2 26.6 

Dare has ascertained with this method that there is a more or less 
constant relationship between the alkalinity of the blood and the 
color index, and he suggests that this may be the reason why the 
results obtained by different investigators differ so widely, as at 
different stages of the disease the color index may change. 

The method is quite convenient and merits the careful attention 
of all laboratory workers. 

Literatuke— v. Jaksch, Zeit. f. klin. Med., 1887, vol. xiii. p. 350. A. Lowy, Arch, 
f. d. gesammte Physiol., 1894, vol. lviii. p. 462. Lowy u. Richter, Deutsch. med. Woch., 
1895, vol. xx. p. 526. Peiper, Arch. f. path. Anat., 1889, vol. cxvi. p. 337. Rumpf, 
Centralbl. f. inn. Med., 1891, vol. xii. p. 447. Kraus, Arch. f. exp. Path. u. Pharmakol., 
vol. xxvi. Engel, Berlin, klin. Woch., 1898, p. 308. Brandenburg, Zeit. f. klin. 
Med., vol. xxxvi. p. 267. Orlowskv, Wratch, 1902, vol. xxii. pp. 1190 and 1222. A. 
Dare, Phila. Med. Jour., Jan. 17, 1903 ; and Johns Hopkins Hospital Bull., July, 1903. 

CHEMICAL EXAMINATION OF THE BLOOD. 

General Chemistry of the Blood. 

A general idea of the chemical composition of the blood may be 
formed from the accompanying table of C. Schmidt, 1 calculated for 
1000 parts : 

1 Cited by v. Gorup-Besanez, Lehrb. d. physiol. Chem., 4th ed., p. 345. 



28 THE BLOOD. 

Man. Woman. 

Corpuscles 513.00 1 389.20 

Water . 349.70 272.60 

Hemoglobin and globulins 159.60 120.10 

Mineral salts 3.70 3.55 

Plasma 486.90 603.80 

Water 439.00 552.00 

Fibrin 3.90 1.91 

Albumins and extractives 39.90 44,79 

Mineral salts 4.14 5.07 

If blood is allowed to flow into a vessel and set aside, it will be 
observed at the expiration of a few minutes that the entire mass has 
become transformed into a semisolid, gelatinous material, which is 
spoken of as the blood-clot or the placenta sanguinis. Still later 
it will be seen that a small amount of straw-colored fluid appears 
on top of the clot, which gradually increases in amount, while the 
clot itself undergoes shrinkage, until finally it floats, greatly dimin- 
ished in size, in the surrounding fluid. The straw-colored fluid 
which has thus been obtained during the process of coagulation is 
spoken of as the blood-serum. 

If a bit of the clot is examined microscopically, it will be seen to 
consist of a more or loss dense network of fibres, the meshes of which 
are filled with blood-corpuscles, which may be washed out, leaving 
the fibrous network, fibrin, behind. 

Chemically speaking, fibrin belongs to the class of the so-called 
coagulated albumins ; it does not occur in the circulating blood, but 
is formed only during the process of coagulation. 

The albumins which are found in the plasma are fibrinogen, serum- 
globulin, and serum-albumin, but while the last two are likewise 
encountered in the serum, the fibrinogen has disappeared, and traces 
of a new albuminous body, fibrin o-globulin, are found. There ap- 
pears to be no doubt that fibrin results from the fibrinogen by a proc- 
ess of dissociation, and that the traces of fibrino-globulin are formed 
at that time. Modern research, furthermore, has shown that the 
transformation of fibrinogen into fibrin is dependent upon the action 
of a special ferment, the fibrin ferment, which is derived in all 
probability from the leucocytes of the blood by a process of plasmo- 
schisis. The presence of serum-globulin apparently hastens coagu- 
lation in an indirect manner, as is done by calcium chloride and the 
calcium salts in general. 

Under normal conditions blood clots in from two to six minutes 
after being shed, while in disease, notably in haemophilia, coagula- 
tion may be greatly retarded or does not occur at all, so that fatal 
hemorrhage may follow the infliction of trifling wounds. A ten- 
dency to hemorrhage is also observed in scurvy, purpura, in some 

1 This figure is too high ; in man it varies between 420 and 470 for 1000 parts of 
blood. 



CHEMICAL EXAMINATION OF THE BLOOD. 



29 



Fig. 3. 



infectious diseases, such as typhoid fever and yellow fever, in 
poisoning with phosphorus, etc. 1 Sicard 2 has pointed out that in 
purpura primary coagulation occurs as with normal blood, but that 
subsequent retraction of the clot and exudation of serum take place 
to only a very limited extent. Normal serum when added to 
fluids, such as hydrocele fluid, which are not spontaneously coagula- 
ble, in the proportion of 1 : 80, induce coagulation in from four to six 
hours. The serum of purpuric patients, on the other hand, is either 
entirely devoid of this property or possesses it to only a very slight 
degree. The addition of a trace of calcium chloride, however, causes 
such serum to behave very much like normal serum. Sicard hence 
suggests that in certain cases of purpura the fibrin ferment, or its 
pro-enzyme is not present in sufficient quantity to cause more than a 
primary coagulation. Subsequent retraction, however, may also be 
due to the action of another variety of fibrin, the zymogen of which 
is absent in purpura. 

Wright's coagulometer may be conveniently employed to deter- 
mine the rapidity of coagulation. The instrument is shown in the 
accompanying illustration (Fig. 3). The essential parts are a tin 
water can, a thermometer registered to about 50° 
C, and a set of eight glass tubes measuring about 
10 cm. in length with a lumen of 0.25 mm. These 
tubes are open at both ends and fit into flannel- 
lined pockets in the leather jacket which surrounds 
the water can. When the instrument is to be 
used, the can is filled with water having a tem- 
perature about that of the body. The tubes are 
slipped into their pockets and remain there until 
they have acquired a similar temperature. They 
are then successively filled about one-half by 
aspiration from a drop of blood obtained from the 
finger or the lobe of the ear at intervals of one 
minute and replaced (properly numbered) in their 
pockets. After two and a half to three minutes 
tube one is examined by attempting to blow its 
contents upon a sheet of paper; if the blood is Wright's coagulometer. 
still liquid, the second tube is examined, then 
the third and fourth, etc., until one is found in which the contents 
have clotted, keeping careful note of the time that has elapsed since 
each was filled. Under normal conditions the coagulation time with 
these tubes will be found to vary between three and five minutes. 
The temperature of the water in the can should be kept uniform 
during the examination by adding hot water if necessary. 




1881. 



Schmidt, PMger's Archiv, vol. xi. pp. 291 and 515. Bojanus, Inaug. Diss., Dorpat, 



2 Sicard, Compt. rend. soc. biolog., vol. li. p. 579. 



30 THE BLOOD. 

The tubes are cleansed by removing the clots with a fine wire ; 
they are then washed with water, with alcohol, and finally with 
ether. 

Since the formation of fibrin begins as soon as the blood has left 
its natural channels, it is apparent that absolutely accurate analyses 
of blood-plasma can hardly be expected. The appended analyses of 
the plasma of the horse's blood are taken from Hoppe-Seyler and 
Hammarsten, the figures having reference to 1000 parts : 

Water 908.4 917.6 

Solids 91.6 82.4 

Total albumins 77.6 69.5 

Fibrin 10.1 6.5 

Globulin 38.4 

Serum-albumin 26.4 

Fat 1.2 1 

Extractives 4.0 ! ., 9 Q 

Soluble salts 6.4 f lzy 

Insoluble salts 1.7 J 

The chief points of difference between plasma and serum are the 
absence of fibrinogen and the presence of traces of fibrino-globulin, 
as well as of large quantities of fibrin ferment, in the latter. 

From the following table it will be seen that a marked difference 
exists in the nature of the mineral ingredients between serum and 
the red corpuscles, the latter being relatively rich in potassium salts 
and phosphorus, and poor in sodium salts and chlorine. The figures 
have reference to 1000 parts of blood : 

Man. Woman. 

Red '"Red 

corpuscles. Serum. corpuscles. Serum. 

K 2 1.586 0.153 1.412 0.200 

Na 2 0.241 1.661 0.648 1:916 

CaO 

MgO 

Fe 2 5 . 

CI 0.898 1.722 0.362 1.440 

PA 0.695 0.071 0.643 2.202 

It is noteworthy that the amount of sodium chloride in the serum, 
6 to 7 pro mille, remains fairly constant no matter whether 
large amounts are ingested or none at all is given. It is probable 
that the sodium chloride of the plasma serves the purpose of pre- 
venting the haemoglobin of the corpuscles from being dissolved by 
the water of the blood. The term " isotonic " has been applied by 
Hamburger 1 to a salt solution which is just strong enough to pre- 
vent the solvent action of the water upon the haemoglobin of the red 

1 Hamburger, Zeit. f. Biol., vol. xxvi. p. 414 ; Ibid., vol. xxvii. p. 259 ; and Vircbow's 
Archiv, vol. cxl. p. 503. 



CHEMICAL EXAMINATION OF THE BLOOD. 31 

» 
corpuscles. In the case of the serum, however, we meet with a 
condition of hyperisotonia — i. e., an amount of salt in excess of that 
actually required in order to prevent the destruction of the red 
corpuscles, the advantage of which is, of course, apparent, if the 
variations to which the amount of water in the blood is subject are 
borne in mind. 

In addition to the substances mentioned, the following are also 
found in the blood : 

Fat occurs in amounts varying from 1 to 7 pro mille in fasting 
animals, while following the ingestion of a meal rich in fats as much 
as 12.5 pro mille have been encountered. 

Soaps, cholesterin, and lecithin have likewise been found. 

Sugar, probably glucose, appears to form a normal constituent of the 
plasma, amounting to from 1 to 1.5 pro mille in man. While it is 
possible to increase this amount to a certain degree by the ingestion 
of large quantities of sugar, this appears in the urine, according to 
Claude Bernard, as soon as 3 pro mille have been exceeded. In addi- 
tion to glucose, another reducing substance has been found in the blood, 
which differs from the former in not being fermentable. According 
to recent researches of P. Mayer, 1 this is in all probability a glucu- 
ronic acid compound. Whether jecorin also occurs in the blood is 
doubtful. 

Among the extractives which have been found, there may be men- 
tioned urea, uric acid, kreatin, carbamic acid, sarcolactic acid, gly- 
cogen, and hippuric acid, and under pathological conditions xanthin, 
hypoxanthin, paraxanthin, adenin, guanin, leucin, tyrosin, lactic acid, 
cellulose, /3-oxybutyric acid, acetone, and biliary constituents. 

It has been pointed out that the color of the blood is referable to 
the presence of haemoglobin in the red corpuscles, and that it varies 
from a bright scarlet-red in the arteries to a dark bluish-red in the 
veins, the exact shade depending upon the amount of oxygen present 
in combination with haemoglobin as oxyhemoglobin. Upon chemical 
examination two other gases may be demonstrated under physiological 
conditions, viz., carbon dioxide and nitrogen. Of these, the latter 
appears to play no part in the body-economy, and the amount present 
merely corresponds to that which would be absorbed by an equal 
volume of distilled water, viz., 1.8 vol. per cent., calculated at 0° C. 
and 760 Hgmm. pressure. 

The amount of oxygen and carbon dioxide, on the other hand, 
undergoes considerable variation, depending upon the particular 
bloodvessel from which the specimen is taken — i. e., whether this be 
an artery or a vein, and, furthermore, upon the velocity of the blood- 
current, the temperature of the body, rest, exercise, etc. 

The relation existing between the amounts of these gases in arteries 
and veins may be seen from the following table : 

1 P. Mayer, Zeit. f. physiol. Chem., vol. xxxii. p. 518. 



32 



THE BLOOD. 



Arterial blood. 

Oxygen 21.6 per cent. 

Carbon dioxide 40.3 

Nitrogen 1.8 



Venous blood. 
6.8 per cent. 
48.0 " 
1.8 " 



Oxygen, as already pointed out, occurs principally in chemical 
combination with haemoglobin (oxyhemoglobin), only 0.26 per cent, 
being present in solution in the plasma. 

Of the carbon dioxide which may be obtained from the blood, 
only one-tenth is held in solution, while the remaining portion is 
found in the red corpuscles, in the form of a loose compound with 
the alkalies of the corpuscles, and possibly also in combination with 
haemoglobin. This portion amounts to about one-third of the total 
quantity, while the remaining two-thirds are probably held in chem- 
ical combination by the alkalies of the plasma and certain albuminous 
bodies. 

The Blood-pigments. 

Haemoglobin and Oxy haemoglobin. — Haemoglobin is a proteid 
in which the albuminous molecule globin is combined with the iron- 
containing pigment hcemochromogen. Upon the presence of the latter 
group depends the great readiness with which haemoglobin forms 
compounds with certain gases, such as oxygen, carbon monoxide, 
carbon dioxide, nitric oxide, and cyanogen. The compound of 
haemochromogen and oxygen is termed haematin ; oxy haemoglobin is 
thus the product of globin and haematin. 

By itself haemoglobin is largely found in the blood of asphyxia. 
Under ordinary conditions it is principally present as oxyhaemo- 
globin ; in arterial blood this preponderates, while in venous blood a 
mixture of both is found. 



Fig. 4. 

Red Orange Yellov) Green 



Cyan-blue 



A a B C _D 

40 50 60 

llliiiiliiiil.iliiliiiilii 





lltlllllllllll 



Spectrum of reduced haemoglobin, (v. Jaksch.) 

On spectroscopic examination haemoglobin in suitable dilution 
shows a single band of absorption between D and E, extending 
slightly beyond D to the left (Fig. 4). 

Oxyhaemoglobin shows two bands of absorption between D and 
E. One band, a, which is not so wide as the second, /9, but darker 
and more sharply denned, borders on D ; the second, Avhich is wider 
but less sharply defined, lies at E (Fig. 5). This spectrum can be 



CHEMICAL EXAMINATION OF THE BLOOD. 



33 



readily transformed into that of haemoglobin by the addition of a 
reducing agent, such as an ammoniacal solution of ferrous tartrate 
(Stokes' fluid), ammonium sulphide, or cuprous salts. 



Fig. 5. 



Bed. Orange 

A 



Yellow 



Green 



Cyan-blue 



a B C 

40 50 




Spectrum of oxyhemoglobin, (v. Jaksch.) 

Under normal conditions the amount of haemoglobin is fairly con- 
stant, but varies somewhat in different countries with the habits of 
the people, the character of the diet, etc. In Germany, as the result 
of sixty-one estimations, Leichtenstern found 14.16 per cent, by 
weight as the average in healthy men, and 13.10 per cent. in. 
women. 

Clinically we express the amount of haemoglobin by relative 
figures as compared with the average normal percentage by weight ; 
on this basis the scale of the various haemoglobinometers is con- 
structed. On these instruments the figure 100 represents the 
average normal value ; this, however, varies somewhat with the 
various forms of haemoglobinometers according to the average per- 
centage by weight which has been taken as a standard in establishing 
the 100 mark. With the Gowers instrument (see page 157) Strauss 
and Rohnstein obtained figures varying between 85 and 125 as nor- 
mal values ; this would furnish an average of 105. Schaumann and 
v. Willebrandt give 88 as the average normal. With the v. Fleischl 
instrument (page 153) I rarely find higher values than 90 per cent, 
in inhabitants of large cities, but with the Dare apparatus (page 151) 
the average results more nearly approach the 100 mark. 

In children the average values are somewhat low T er than in the 
adult. Stierlin gives 79.7 per cent, for boys and 82.1 for girls. 
Borchmann's values are even lower, viz., 55 and 80 ; Gundobin gives 
70 and 95. 

The ingestion of large amounts of water does not cause a dilution 
of the blood and hence a diminution of the amount of haemoglobin ; 
but relatively higher values are found upon the withdrawal of liquids, 
owing to a concentration of the blood as a whole. Fat persons show 
smaller values than correspond to their age. 

A pathological decrease in the amount of haemoglobin is spoken 
of as oligochromcemia, and is observed in all forms of anaemia from 
whatever cause. 

The lowest values are found in chlorosis, in which the oligochromae- 
3 



34 THE BLOOD. 

mia far exceeds the oligocythcemia, viz., the diminution in the number 
of the red cells (see page 59). In an analysis of 94 cases I found 
an average of 42.5 per cent. ; the lowest value was 17.5 (Fleischl). 
There are instances on record, however, in which the reading has 
been even lower. 

Very low figures are also seen in splenic anaemia, and it is rare, 
excepting in chlorosis, to find such a low grade of chromaemia 
associated with a blood-count which is normal or may indeed be 
above normal. The average of 13 estimations given by Osier was 
47 per cent. 

In pernicious anaemia the oligocythemia exceeds the oligochro- 
maemia. The loss of haemoglobin is, however, also quite marked, 
and may be as great as in the most extreme cases of chlorosis. In 
the series of 23 cases collected by Strauss and Rohnstein the average 
value was 25 per cent. (Gowers) ; in 9 cases it was lower than 20 
per cent. 

In the early stages of leukaemia the loss of haemoglobin is often 
not especially marked ; later the anaemia may become quite intense, 
but it is to be noted that the oligochromaemia is not necessarily of 
high grade even in well-developed cases. Ehrlich thus cites cases in 
which the Gowers instrument gave readings of from 60 to 70 per 
cent. On the other hand, there are cases in which the oligo- 
chromaemia is an early feature of the disease, and in one instance of 
this kind I obtained a reading of only 27 per cent. 

While in typhoid fever the amount of haemoglobin is always 
reduced (Osier), and usually to a greater extent than the number of 
the red corpuscles, the most severe grades of anaemia may here be 
encountered during convalescence, when the amount of haemoglobin 
may fall to 20 per cent. 

In the early stages of carcinoma of the stomach the cachexia is 
not well pronounced. Sclmle states that in his analysis of 198 cases 
it occurred in only 30 per cent. Later, however, the loss of haemo- 
globin is quite marked ; the values may indeed approach those seen 
in chlorosis and pernicious anaemia. 

An intense grade of anaemia is produced in cases of generalized 
septicaemia, and as Ewing remarks no form of the acute disease 
appears to act more violently than does puerperal or uterine sepsis. 
A diminution in the amount of haemoglobin to 20 per cent, is here 
not uncommon. In the chronic cases also a high grade of oligo- 
chromaemia is a constant feature. In a case of lumbar abscess of 
six months' duration I found 21 per cent, of haemoglobin, with 
1,025,000 red cells. The haemoglobin in all these cases diminishes 
more rapidly than the number of the red cells. 

In pulmonary tuberculosis a diminution in the amount of haemo- 
globin is seen essentially in the third stage of the disease (40-45 per 
cent.), while previously fairly normal values are obtained (90-95 



CHEMICAL EXAMINATION OF THE BLOOD. 35 

per cent.). It is to be noted, however, that a certain grade of 
anaemia (69 per cent.) is quite commonly observed, even in the first 
stage, in those cases in which the disease has been of very gradual 
onset, viz., in patients who often have suffered from tubercular 
affections (scrofula) since childhood. In the third stage the anaemia 
is well marked (40—50 per cent.) (Appelbaum). 

A notable diminution in the amount of haemoglobin is observed in 
chronic nephritis, chronic enteritis, iu chronic lead and mercurial 
poisoning, in syphilis, etc. 

In syphilis the anaemia develops at a time when the entire organism 
has been thoroughly infected. The lowest haemoglobin values are 
reached just before or coincidently with the appearance of the rash. 
In the secondary stage the degree of oligochromaemia, cceteris paribus, 
may be regarded as a fair index of the severity of the infection. In 
untreated cases the haemoglobin remains low for several days or even 
for weeks. A gradual rise then occurs which is associated with 
beginning involution of the exanthem. In uncomplicated cases 
normal values may subsequently be reached even without treat- 
ment ; a fall again occurs with relapses. Similar changes are 
observed in the tertiary stage. Especially interesting are the obser- 
vations of Justus on the blood-changes which occur in the course of 
mercurial treatment ; Justus ascertained that a rapid and material 
diminution of the haemoglobin (10-20 per cent.) occurs when a large 
(medicinal) amount of mercury is introduced at one time into the 
body of the infected individual. This decrease is only observed in 
the blood of patients with florid syphilis ; it is specific and does not 
occur in healthy nor in otherwise diseased individuals. The reaction 
is demonstrable in every form of syphilitic infection (secondary, 
tertiary, and hereditary) as soon as the more distant lymph-glands 
begin to swell. It disappears, or is at least no longer demonstrable 
with beginning involution of the symptoms. 

Justus' Syphilitic Blood-test. — A haemoglobin estimation should be 
made on two consecutive evenings at the same hour ; on the second 
evening an inunction is given of not less than 3 grammes of the 
officinal gray ointment in the case of adults, or of 1 gramme in the 
case of children. The characteristic drop will then be demonstrable 
in the course of the following forenoon ; examinations should, if 
necessary, be made at intervals of one or two hours. Justus regards 
a drop of more than 5 per cent, as evidence of the existence of florid 
syphilis. His conclusions regarding the diagnostic value of the 
test are based upon a study of 500 cases. His results have in the 
main been confirmed, but it is necessary to follow the directions just 
outlined, as the drop may otherwise be overlooked. 

During anaesthesia by ether the amount of haemoglobin is always 
absolutely reduced. In some instances there is an apparent increase, 
but this is never proportionate to the rise in the number of the red 



36 THE BLOOD. 

cells which is simultaneously observed (Da Costa, Kalteyer). Owing 
to the haemocytolysis which thus undoubtedly takes place a very low 
percentage of haemoglobin should be regarded as a counterindication 
to general anaesthesia. A lower value than 50 per cent, is now 
regarded by many as a dangerous figure. 

For the estimation of haemoglobin see page 150. 

Literature. — Strauss u. Rohnstein, Die Blutzusarnniensetzung b. d. verschied- 
eiien Anaemien, Hirschwald, Berlin, 1901. Appelbaum, Berl. klin. Woch., 1901, vol. 
xxxix. p. 7. Quincke, " Zur Pathologie d. Blutes," Deutsch. Arch. f. klin. Med., 
vols. xxv. and xxvii. Leichtenstern, Unters. iiber d. ILenioglobingehalt d. Blutes im 
gesunden u. kranken Zustande, Leipzig, 1878. W. Osier, "On Splenic Anaemia," 
Am. Jour. Med. Sci., 1902, vol. cxxiv. p. 763. Justus, Virchow's Archiv, vol. cxl. 
p. 1 ; and Deutsch. Arch. f. klin. Med., 1902, vol. lxxv. p. 1. 

Haemoglobinsemia. — The term haemoglobinaemia has been applied 
to a condition in which the haemoglobin is dissolved out from the red 
corpuscles, and, appearing in the plasma as such, leads at first to a 
very decided choluria and in extreme cases to haemoglobinuria. 

Various poisons, such as potassium chlorate, carbolic acid, pyro- 
gallic acid, naphtol, arsenic, sulphide of antimony, hydrochloric 
acid, sulphuric acid, antifebrin, antipyrin, phenacetin, sulphonal, 
tincture of iodine, when given hypodermically, or even internally in 
sufficiently large doses, will call forth a haemoglobinaemia which is 
followed by haemoglobinuria. 

Fresh morels also contain a poison which is capable of producing 
an intense haemoglobinuria, and which may be extracted with hot 
water. 

In acute and chronic infectious diseases of a severe type, such as 
scarlatina, typhoid fever, intermittent fever, icterus gravis, syphilis, 
as also in diseases depending upon a hemorrhagic diathesis, such as 
variola haemorrhagica, scurvy, as also following insolation, extensive 
burns, and frostbite, haemoglobinaemia, leading to haemoglobinuria, is 
not infrequently observed. In syphilis a moderate grade of haemo- 
globinaemia can be demonstrated by spectroscopic examination of 
the serum within two or three minutes following an intravenous in- 
jection of mercuric chloride in medicinal doses. (See also Justus' 
test.) 

An epidemic haemoglobinuria of the newly born and a paroxysmal 
or intermittent haemoglobinuria, both of unknown origin, have like- 
wise been described. 

In a case of Raynaud's disease which I had occasion to observe in 
the clinic of Dr. H. M. Thomas, at the Johns Hopkins Hospital, 
haemoglobinuria at times followed epileptiform seizures. 

Haemoglobinaemia followed by haemoglobinuria is finally observed 
after transfusion of the blood of one mammal into the circulation of 
another. 

In some cases, and particularly in those following poisoning with 



CHEMICAL EXAMINATION OF THE BLOOD. 



37 



chlorates, etc., the hemoglobinemia ultimately leads to a well-pro- 
nounced methemoglobinemia (see below). 

A hemoglobinemia, aside from the urinary examination, may be 
readily recognized by a spectroscopic examination of the serum, when 
the two bands of absorption of oxyhemoglobin will be observed. 

A very simple method which may be employed for the same pur- 
pose is the following : a small amount of blood is drawn from the 
patient by means of cupping-glasses and immediately placed on ice ; 
where it is allowed to remain for from twenty to twenty-four hours. 
At the expiration of this time the clot will have shrunk, floating, if 
the blood is normal, in the clear, straw-colored serum, while a 
beautiful ruby-red color is obtained in cases of henioglobinemia. 
If some of this serum is then heated to a temperature of from 70° 
to 80° C, the coagulum in the presence of hemoglobin will present 
a more or less deep-brown color. 

Literature.— Ponfick, Verhandl. d. Cong. f. inn. Med., 1883, vol. ii. p. 205. 

Stadelmann, Arch. f. exp. Path. u. Pharmakol., 1882, vol. xv. p. 337, and 1884, 

vol. xvi. pp. 118 and 221. Afanassiew, Zeit. f. klin. Med., 1883, vol. vi. p. 281. 
v. Jaksch, Verhandl. d. Cong. f. inn. Med., 1891, vol. x. p. 353. 

Carbon Monoxide Haemoglobin. — In cases of coal-gas poisoning 
the blood, both of arteries and veins, presents a bright cherry-red 
color, owing to the presence of carbon monoxide hemoglobin. 

Such blood, when properly diluted, like oxyhemoglobin, shows two 
bands of absorption between D and E (Fig. 6), which are nearer the 
violet end of the spectrum, however, and may readily be distinguished 
from those referable to oxyhemoglobin by the addition of a reducing 
agent. This will not affect the spectrum of carbon monoxide hemo- 
globin, while that of oxyhemoglobin is transformed into the spectrum 
of reduced hemoglobin. 

For medico-legal purposes a number of additional tests have been 
devised, among which that suggested by Hoppe-Seyler is one of the 
simplest and at the same time most reliable. The blood is treated 
with double its volume of a solution of sodium hydrate (sp. gr. 



Cyan-blue 




Spectrum of carbon monoxide haemoglobin, (v. Jaksch.) 



1.3). Normal blood is thus changed into a dirty-brownish mass, 
which exhibits a trace of green when spread upon a porcelain plate, 
w r hile carbon monoxide blood yields a beautiful red under the same 
conditions. 



38 



THE BLOOD. 



Nitric Oxide Haemoglobin. — The blood in cases of poisoning 
with nitric oxide, owing to the presence of nitric oxide haemoglobin, 
yields a spectrum which is similar to that of carbon monoxide haemo- 
globin ; the bands, however, are less sharply defined and paler than 
those of the latter, and, like these, do not disappear on the addition 
of a reducing substance. 

Hydrogen Sulphide Haemoglobin (Methaemoglobin Sulphide). 
— In cases of poisoning with hydrogen sulphide no definite changes 
can be discovered in the blood upon spectroscopic examination, 
although Hoppe-Seyler has shown that haemoglobin may enter into 
combination with this gas. It is stated, however, that in such cases 
the blood becomes dark and of a dull-greenish tint, and that the 
distinction between arterial and venous blood is lost. 

Carbon Dioxide Haemoglobin. — With carbon dioxide, as men- 
tioned above, haemoglobin is also thought to enter into combination, 
the spectrum being similar to that of reduced haemoglobin. The 
latter, in fact, is formed artificially when carbon dioxide is passed 
through a solution of oyxhaemoglobin. If this process is carried 
further, the haemoglobin is decomposed and globin is thrown down ; 
an absorption-band is then obtained which is similar to that result- 
ing when haemoglobin is decomposed with acids (see below), and is 
no doubt referable to the presence of free haemochromogen. 

Of the blood-changes occurring in cases of poisoning with hydro- 
cyanic acid and acetylene, but little is known, and the reader is 
referred to works on toxicology for their consideration. 

Haematin. — If oxyhaemoglobin in aqueous solution is heated to a 
temperature of from 60° to 70° C, it is decomposed into globin and 
haematin. The same result is reached by treating the aqueous solu- 
tion with acids, alkalies, or the salts of various heavy metals. 

Haematin is an amorphous, blackish-brown or bluish-black sub- 
stance which is frequently encountered in old transudates, in the 
stools after hemorrhages, and after meals consisting largely of red 
meats. It is said to occur in the urine in cases of poisoning with 
arsenic, and in the blood of animals poisoned with nitrobenzol its 
presence can likewise be demonstrated with the spectroscope. 



Fig. 7. 



Yellow 



Green 



Cyan-blue 



B 

40 50 

Mi l 11 , 111 1 1,1 1 III I l„ 



D 

60 

ill I I 







100 

llllll lllll III 



Spectrum of haematin in alkaline solution, (v. Jaksch.) 



In acid solution it shows a well-defined spectral band between 
C and D (Fig. 9). Between D and F a second band is seen, which 



PLATE I. 




Hsemin Crystals. 



CHEMICAL EXAMINATION OF THE BLOOD. 



39 



is much wider but less sharply defiued thau the first, and may be 
resolved into two bands by dilution, one between b and F 3 near F, 
and another between D and F, near F ; a faint fourth band may 
also be seen between D and F, near D. As a rule only the two 
bands between D and F are visible. 



Fig. 8. 



Red Orange 



Yellow 



Gh-een 



Cyan-blue 



a 



B C 

±0 50 

■ ■InmI.m.LmI. 



Eb F 

80 90 100 

mWWllluIiillliJ 



Spectrum of reduced hsematin. (v. Jaksch.) 

In alkaline solutions it shows but one broad band, the greater 
portion of which lies between C and D, extending slightly beyond 
D (Fig. 7). 

If an alkaline solution of hsematin is treated with a reducing 
substance, reduced hsematin (hsemochromogen) results, which gives 
rise to two absorption bands between D and E (Fig. 8). 

Haemin. — Hsematin readily combines with one molecule of 
hydrochloric acid to form hsemin. This substance crystallizes in 
light- or dark-brown rhombic plates or columns, which are quite 
characteristic (Plate I.). They bear the name of their discoverer, 
Teichmann. The size of these crystals varies with the manner in 
which they are produced, the largest specimens being met with 
when the glacial acetic acid (see below) is allowed to evaporate as 
slowly as possible. Specimens measuring from 15 [i to 18 fi in 
length may then be seen. Smaller crystals will be present at the 
same time, occurring either singly or in the form of stars, rosettes, 
and crosses. 

As these crystals may be obtained from mere traces of blood, their 
formation must be regarded as conclusive evidence in medico-legal 
examinations. Lewin and Rosenstein have pointed out, however, 
that under certain conditions a negative result may be reached, even 
if the coloring-matter is derived from the blood. This is the case 
especially when the hsemoglobin has been transformed into hsemo- 
chromogen or hsematoporphyrin, or when substances have been mixed 
with the blood which are either capable of altering its general com- 
position or w r hich, through their mere presence, interfere with the 
reaction. Such substances are certain salts of iron (rust), lead, mer- 
cury, and silver ; further, lime, animal charcoal, and sand, when 
intimately mixed with the blood. In medico-legal cases a spectro- 
scopic examination should hence also be made whenever the hsemin 
reaction is not obtained. 

Method. — A small drop of normal salt solution is carefully 



40 THE BLOOD. 

evaporated on a slide, when a few particles of the suspected material, 
powdered or teased as finely as possible, are placed on the delicate 
layer of crystallized salt. Glacial acetic acid is now added drop by 
drop and the specimen carefully heated (three-quarters to one minute) 
until bubbles begin to form. While evaporation is being continued 
glacial acetic acid is further added until a light-brown tint appears. 
As soon as this point is reached, the last traces of the acid are 
allowed to evaporate, the specimen being held at a greater distance 
from the flame. A drop of glycerin is then added and the prepara- 
tion covered with a cover-glass. The examination is made with a 
one-fifth or a one-sixth objective. Attention is especially directed 
to brownish streaks or specks, which, in the presence of blood, can 
usually be made out Avith the naked eye. 

Methsemoglobin. — Methemoglobin is a pigment closely related to 
oxyhemoglobin, and is frequently encountered in hemorrhagic transu- 
dates, cystic fluids, and in the urine in cases of hematuria and hemo- 
globinuria. In the circulating blood methsemoglobin is found after 
the ingestion of large quantities of potassium chlorate, notably so in 
children, as also after the inhalation of nitrite of amyl, the use of 
kairin, thallin, hydrochinon, pyrocatechin, iodine, bromine, turpen- 
tine, ether, perosmic acid, permanganate of potassium, and antifebrin 
(see Hemoglobinemia). 

Fig. 9. 

Red Orange Yellow Green Cyan-blue 

A a B C D Eb F 

40 50 , 60 70 _ 80 90 100 110 

lllllllll lllll 



LLLI 



LLL 



mi 



Spectrum of methaemoglobin in acid and neutral solutions, (v. Jaksch.) 

The spectrum of an aqueous or slightly acidified solution of methse- 
moglobin (Fig. 9) closely resembles that of an acid solution of 
hematin, but differs from this in the ease with which it is trans- 
formed into that of hemoglobin when an alkali and a reducing 
substance are added. The spectrum of hematin under the same 
conditions is transformed into that of an alkaline solution of hemo- 
chromogen. In alkaline solutions, on the other hand, two bands 
of absorption are observed, which are similar to those of oxy- 
hemoglobin, but differ from these in the fact that the band nearer 
E, /9, is more pronounced than the one at D, a. A third, but 
very faint, band may further be observed between C and D y 
near D. 

Hesmatoidin. — Small amorphous particles of an orange or ruby- 
red color, or crystals belonging to the rhombic system, occurring 
either singly or in groups, are frequently met with in the sputum, 



CHEMICAL EXAMINATION OF THE BLOOD. 



41 



the urine, and the feces, as well as in old extravasations of blood. 
Thev were discovered by Yirehow, who applied the term hsematoidin 
to this particular pigment, the hseinic origin of which is undoubted, 
It is supposed! v identical with bilirubin. 



Fig. 10. 



Bed Orange 



Green 



Cyan-blue 




ml 


Mil 


Mil! 


...L.L 


LI. J, 


■Uiifl- 


I....I....L... 












.3 ■ '" ■ ■ i 





Spectrum of hsematoporphyrin in alkaline solution. 



Haematoporphyrin. — Hsematoporphyrin is likewise a derivative 
of hsematin, and, according to Nencki and Sieber, isomeric with 
bilirubin. In dilute solution with sodium carbonate it shows four 
bands of absorption, one between C and D, a second one, broader 
than the first, about D, especially marked between D and E, a third 
one, not so broad and less sharply defined between D and E, and a 
fourth one, broad and dark, between b and F (Fig. 10). 

The clinical significance of this body, which also appears in the 
urine, as well as the causes which give rise to its formation, are as 
yet unknown (see Hsematoporphyrinuria). It has been found post 
mortem in the blood, in a case of sulphonal poisoning, by Taylor 
and Sailer. 1 

While it is usually possible, as pointed out above, to recognize 
definitely the presence of blood by the haemin test, recourse should 
always be had to a spectroscopic examination whenever the exact 
nature of the pigment under consideration is to be determined. 

The Spectroscope. — The spectroscope (Fig. 11) essentially con- 
sists of a tube (A), provided with a slit at its distal 'end, which may 
be narrowed or widened, and a collecting-lens at its proximal end. 
Through the latter, rays of sunlight or of artificial light are thrown 
upon a prism (P), where they are decomposed into a colored spec- 
trum, which is viewed through an astronomical telescope (B). 
Through a third tube (C) a fine scale, illuminated by artificial light, 
is reflected by the prism to the eye of the observer, appearing im- 
mediately above the colored spectrum. The left of this is red, 
passing into yellow, this into green, then into blue, indigo, and finally 
into violet, which occupies the right end. These colors, however, 
are not continuous, but are interrupted by a large number of verti- 
cally placed dark lines, named after Frauenhofer. The most marked 

1 A. E. Taylor and J. Sailer, Contrib. from the William Pepper Laboratory, Pbila., 
1900, p. 120. 



42 



THE BLOOD. 



of these are designated by the letters A, a, B, C, B, E, b, F, G, 
and H. Of these, A is found at the left end and B in the middle 



Fig. 11. 




The spectroscope. (Neubaueb.) 



of the red portion of the spectrum, C at the boundary of the red and 
the orange, D in the yellow, E in the green, F in the blue, G in the 
indigo, and H in the violet portion ; a is situated in the red between 

Fig. 12. 





Browning's spectroscope. (Zeiss.) 



A and B, nearer A, and b in the green between E and F, nearer 
E (see Fig. 4). 

If now a colored medium is placed between the slit and the light, 
not all the ravs of colored light reach the eye, but some become ab- 



THE PROTEIDS OF THE BLOOD. 43 

sorbed. In the case of the blood, for example, it may thus be seen 
that a portion of the yellow and a portion of the red rays are ab- 
sorbed, a spectrum of this kind being spoken of as an absorption- 
spectrum. 

For clinical purposes various instruments, modifications of the one 
described, have been devised, among which those of Desego, of 
Heidelberg, Zeiss, of Jena (Fig. 12), and Hoffman, of Paris, as well 
as Hering's lensless spectroscope, and Plenocque's instrument, are 
quite serviceable. 

THE PROTEIDS OF THE BLOOD. 

In considering the proteids of the blood from a clinical point of 
view, it is necessary to distinguish between an increase and a dimi- 
nution in their normal amount, constituting the conditions of hyper- 
albuminosis and hypalbuminosis, respectively. As may be expected, 
the former is met with whenever water is more rapidly withdrawn 
from the system than it can be supplied, and is hence observed in 
cases of cholera, acute diarrhoea, following the use of purgatives, etc. 
This increase in the amount of proteids is only a relative increase, 
however. The occurrence of an absolute increase has not been 
satisfactorily demonstrated. An absolute hypalbuminosis, on the 
other hand, is observed following a direct loss of proteids from 
the blood, as in hemorrhage, dysentery, albuminuria of high degree, 
the formation of large collections of pus, etc. This is generally 
associated with a relative increase in the amount of water — i. e., a 
hydraemia — which is particularly noticeable after hemorrhages, and 
referable to a diminished secretion and excretion of water, as well 
as to a direct absorption from the tissues. Hypalbuminosis has also 
been observed in pernicious anaemia, and is dependent partly upon 
a diminution in the amount of the albumins of the serum and partly 
upon a decrease in the weight of the corpuscular solids. The amount 
of serum-albumin is about normal, while the globulins are much 
diminished. 1 

The term hyperinosis has been applied to a condition in which the 
amount of fibrin is increased. This is said to occur in various 
inflammatory diseases, such as pneumonia, pleurisy, acute articular 
rheumatism, and erysipelas, while a diminished amount of fibrin, 
hypinosis, has been observed in malaria, nephritis, pyaemia, and per- 
nicious anaemia. 

In order to determine the amount of fibrin, 30 to 40 c.c. of blood, 
obtained by means of cupping-glasses, are placed in a previously 
weighed beaker, fitted with an India-rubber cap, through the centre 
of which passes a piece of whalebone, firmly fixed. The blood is 
defibrinated by beating with the whalebone, when the beaker with 
its contents is weighed, the difference indicating the weight of the 

1 Erben, Zeit. f. klin. Med., 1900, vol. xl. p. 266. 



44 THE BLOOD. 

blood. The beaker is then filled with water and the mixture again 
beaten. The fibrin is allowed to settle and after being washed 
with normal salt-solution filtered through a filter of known weight. 
It is further washed with normal salt solution until free from color- 
ing-matter, then boiled in alcohol to dissolve out the fat, cholesterin, 
and lecithin, dried at 110° to 120° C, and on cooling weighed over 
sulphuric acid. 

In leukemic blood v. Jaksch l was able to demonstrate peptones 
in considerable quantities, and especially so after death, when the 
amount progressively increased as decomposition advanced. Mat- 
thes, 2 on the other hand, could detect no true peptones, but found 
that the blood contained a deutero-albumose. In one case the serum 
contained an abundance of nucleo-albumin, derived in all probability 
from degenerated leucocytes. 

More recently album oses have also been found in a case of abscess 
of the brain associated with albumosuria. Freund 3 claims that 
peptones are found in the blood in cases of sarcoma, while in carci- 
noma they are absent. This statement, however, lacks confirmation. 

Following the injection of nuclein and spermin, moreover, albu- 
rnossemia appears to occur quite constantly both during the stage of 
hypo- as well as hyperleucocytosis. After injections of pilocarpin 
albumosuria is observed only in association with hyperleucocytosis. 

In order to test for albumoses, all other proteids should first be 
removed, when a positive biuret-reaction in the filtrate will indicate 
their presence (see also Salkowski's test). 

Carbohydrates. 

Sugar. — Sugar, as indicated above, is a normal constituent of the 
blood, its quantity varying between 1 and 1.5 pro mille. Under 
pathological conditions this amount may be exceeded by far, and 
notably so in diabetes, in which Hoppe-Seyler found as much as 
9 pro mille in a given case. 

In addition to sugar, a non-fermentable reducing substance has 
been encountered in the blood, which, according to Mayer's recent 
investigations, appears to be a compound glucuronate. 4 The presence 
of jecorin in the blood still remains to be proved. 

Large quantities of a reducing substance, the greater portion of 
which consisted of sugar, have been met with by Trinkler in carci- 
noma ; it was observed at the same time that carcinoma of the inter- 
nal organs w T as associated with far greater amounts of sugar than 
cancerous disease of the skin and the mucous membranes. It is 
also interesting to note in this connection that an increase in the 

1 v. Jaksch, Zeit. f. physiol. Chem., vol. xvi. p. 243. 

2 Matthes, Berlin, klin. Woch., 1894, Nos. 23 and 24. 

3 Freund u. Obermayer, Zeit. f. physiol. Chem., vol. xv. p. 310. 

4 P. Mayer, Ibid., vol. xxix. p. 59. 



THE PROTEIDS OF TEE BLOOD. 45 

degree of the cachexia was not accompanied by an increase in the 
percentage of sugar. 

The results reached by Trinkler 1 apparently also bear out the 
correctness of the conclusions formed by Freund, who claimed that 
a differential diagnosis between carcinoma and sarcoma, in which 
latter condition no increase in the amount of sugar was noted, can 
always be effected upon the basis of an examination of the blood 
in this direction. 

In the following table the percentages found in the different dis- 
eases investigated are given, from which it is apparent that, next to 
carcinoma, the largest quantities of sugar are met with in the infec- 
tious diseases and the lowest figures in diseases of the kidneys : 

Average. Minimum. Maximum. 
Per cent. Per cent. Per cent. 

Carcinoma 0.1819 0.1023 0-3030 

Typhoid fever 0.0950 0.0875 0.1022 

Pneumonia 00943 0.0813 0.1092 

Dysentery 0.0838 0.0796 0.0915 

Heart disease 0.0737 0.0664 0.0897 

Peritonitis 0.0701 0.0450 0.0917 

Tuberculosis 0.0653 0450 0.0817 

Syphilis 0.0553 0.0449 0.0748 

Nephritis and uraemia .... 0.0489 0.0321 0.0559 

In order to demonstrate sugar in the blood, 15 to 30 grammes, 
obtained by venesection or cupping-glasses, are placed in an evapo- 
rating-dish and treated with an equal weight of finely powdered 
sodium sulphate and a few drops of acetic acid. The mixture is 
brought to the boiling-point and passed through a muslin filter as 
soon as the coagulum has become black and spongy, water having 
previously been added to the original volume. The nitrate is passed 
through Swedish paper. In the final nitrate the sugar is then esti- 
mated as described elsewhere (see Urine). 

Or, the blood is treated with four or five times its volume of 
alcohol (94 to 96 per cent.) slightly acidified with acetic acid. 
The mixture is allowed to stand for several hours, no heat beiug 
applied. It is then filtered and evaporated on a water-bath until all 
the alcohol has been driven off. Should any albumin separate out 
during this process, the residue is again extracted with alcohol. The 
final residue is dissolved in water. In this solution the sugar is then 
estimated according to Knapp's method. 

Of late, Cavazzani has drawn attention to another method of free- 
ing the blood from proteids, which is said to be entirely satisfactory 
and less expensive. To this end, 20 to 30 c.c. of blood are added 
to 200 c.c. of distilled water in a porcelain dish and treated with 
five or six drops of a solution consisting of 10 parts of acetic acid 
(sp. gr. 1.040) and 1 part of lactic acid. The mixture is boiled for 
eight to ten minutes, filtered, and the coagulum washed repeatedly 

1 Trinkler, Centralbl. f. d. med. Wiss., 1890, p. 498. Freund u. Oberniayer, loc. cit. 



46 THE BLOOD. 

with hot water and finally pressed out in a piece of muslin. The 
resulting filtrates, which are practically colorless, are then concen- 
trated to a small volume, and any traces of albumin, which may still 
separate out, filtered off. If an excess of the acid solution has been 
added, it may happen that the mixture does not clear up on boiling. 
It is then only necessary to add a few crystals of sodium carbonate, 
when coagulation will occur at once. On the other hand, it may at 
times be necessary to add a few more drops of the acetic acid 
solution. 

Williamson's Diabetic Blood Test. — This test is of much interest, 
and may possibly serve to differentiate the ordinary forms of diabetes 
from that in which the blood-sugar is not increased. It is based 
upon the observation that a warm alkaline solution of methylene- 
blue is decolorized by grape-sugar. As with Bremer's test (see page 
63), a positive result may at times be obtained, when the sugar has 
temporarily disappeared from the urine. 1 

Method. — Twenty cbmm. of blood, obtained from the finger or 
the ear, are carefully measured off with the aid of the capillary 
pipette which accompanies Grower's hsemocytometer, and are mixed in 
a test-tube of small calibre with 40 cbmm. of distilled water. To this 
mixture 1 c.c. of an aqueous solution of methylene-blue (1 : 6000) 
and 40 cbmm. of a 6 per cent, aqueous solution of potassium 
hydrate are added. A control-tube is similarly charged with non- 
diabetic blood. The two specimens are then placed in boiling water 
and allowed to remain for from three to four minutes, without 
shaking. At the end of this time it will be seen that the diabetic 
blood has decolorized the methylene-blue solution, which has turned 
a dirty yellowish-green or yellow, while the non-diabetic specimen 
has retained its original color. 

The quantity of blood used should not exceed the amount indi- 
cated, as a decolorization of the methylene-blue also results with 
non-diabetic blood if large amounts, such as 60 cbmm., are em- 
ployed. 

The reaction is supposedly due to an increase of glucose in the 
blood, and was obtained in all of forty-three cases of diabetes which 
were examined. It is said to be obtainable for a considerable time 
after death. Adler 2 found the reaction in all of nine cases of dia- 
betes, while in one hundred and twenty-one non-diabetic cases nega- 
tive results were reached. Very curiously, it was absent in non- 
diabetic glucosurias. Adler believes the reaction to be referable to 
a diminished alkalinity of the blood. 

Glycogen. — There appears to be no doubt that glycogen normally 
occurs in the blood of various animals. Huppert 3 succeeded in 

1 E. T. Williamson, Centralbl. f. inn. Med., vol. xviii. No. 33. 

2 Adler, Zeit. f. Heilk., 1900, vol. xxi. No. 11. 

3 Huppert, Zeit. f. physiol. Chem., 1893, vol. xviii. p. 144. 



THE PROTEIDS OF THE BLOOD. 47 

demonstrating its presence in all animals examined, the amount vary- 
ing between 0.114 and 1.560 grammes for 100 parts of blood (see 
also Iodophilia, page 83). 

Cellulose. — Cellulose has occasionally been found in the blood of 
tubercular patients. 

Urea. 

Urea occurs normally in the blood in traces — 0.016 to 0.020 per 
cent. Larger amounts are encountered whenever, for any reason, as 
in nephritis, various diseases of the urinary organs, cholera Asiatica, 
cholera infantum, eclampsia, etc., its elimination is impeded, or 
whenever, as in fever, owing to increased albuminous decomposition, 
urea i& formed in abnormally large quantities. 

In this connection it is interesting to note that a smaller amount 
of urea is found in fatal cases of eclampsia than in those ending in 
recovery, an observation which has been explained by the assumption 
that in this condition the functional activity, not only of the kidneys, 
but also of the liver, is lost. 

The methods which are available for the detection of urea in the 
blood are still too complicated for clinical purposes, and the value 
of the information derived so small as hardly to warrant the labor 
involved. Hoppe-Seyler's method should be employed whenever an 
examination in this direction is deemed advisable. 1 

Uraemia. — Formerly, it was thought that the complex of symp- 
toms generally spoken of as uraemia was referable to the retention 
in the blood of urea or ammonium carbonate. This view has since 
been disproved, however, although it must be admitted that in 
uraemia an increased amount of urea is frequently noted. Other 
views, according to which uraemia is referable to an accumulation 
of potassium salts, of extractives, and especially of kreatinin, or of 
ptomai'ns in the blood, must still be regarded as being sub judice. 
There is no reason, however, to ascribe the uraemic condition to the 
retention in the blood of one particular constituent of the urine, and 
it is not improbable that a retention of all may be responsible for 
the symptoms observed. 

Literature. — Feltz and Bitter, Del'uremie exper., Paris, 1881. Astaschewsky, 
St. Petersburg, med. Woch., 1881, No. 27. Bouchard, Lecons sur 1' autointoxication, 
Paris, 1887. Bovighi, Eivista clinica, 1886. 

Ammonia. 

Normal venous blood, according to the researches of Winterberg, 
contains about 1 mgrm. of ammonia for each 100 c.c. In febrile 
conditions variable results are obtained, but it appears certain that 
a definite relation between the height of the fever and the amount of 

1 See Hoppe-Seyler, Handbuch der physiologisch- und patkologisck-cueniischen 
Analyse, Vierte Auflage, p. 363, 



48 THE BLOOD. 

ammonia does not exist. In chronic hepatic diseases, and notably 
in cirrhosis, it is not increased. The course of acute yellow atrophy 
also is not necessarily associated with an increase. Very significant 
is the observation that in uraemia following extirpation of the kid- 
neys no increase is observed. An ammonisemia in the sense of v. 
Jaksch can hence scarcely be said to exist. 

Litek.vtu:re. — Nencki, Pawlow, and Zaleski, Arch. f. exp. Path. u. Pharmakol., 
1896, vol. xxxvii. p. 26. Winterberg, Wien. klin. Wocb., 1897, p. 330. 

Uric Acid and the Xanthin-bases. 

Uric Acid. — Formerly, the presence of appreciable amounts of 
uric acid in the blood was regarded as pathognomonic of gout. But 
we now know that a lithsemic condition may occur also in other 
diseases. Traces of uric acid are indeed encountered under normal 
conditions. 

A definite lithsemia has been observed in a variety of disorders, 
such as pneumonia, acute and chronic nephritis, chronic gastritis, 
catarrhal angina, conditions associated with an insufficient aeration 
of the blood, as in the various diseases of the heart, in pleurisy with 
exudation, emphysema when accompanied by cyanosis, the severer 
forms of anaemia, etc. v. Jaksch claims to have found uric acid in 
the blood in 88.88 per cent, of his cases of nephritis. Fever in 
itself does not appear to lead to an increased production of uric acid, 
as negative results were obtained in nine cases of typhoid fever out 
of eleven, in five cases of acute articular rheumatism out of six, etc. 
The conclusion is thus forced upon us that the diminished alkalinity 
of the blood observed in nephritis and anaemia is, to some extent at 
least, dependent upon the presence of a nitrogenous acid, while the 
diminished alkalinity of the blood observed in fevers is not referable 
to this cause. 

From a survey of the literature upon the subject it appears that 
an increased elimination of uric acid in the urine is not necessarily 
accompanied by an increase in the amount of uric acid in the blood. 
Further researches in this direction are, however, highly desirable, 
and particularly so in connection with the various forms of gastric 
disease, in which an increased elimination of uric acid, according 
to my experience, is so frequently observed. 

The assumption that acute attacks of gout are referable to an 
increased alkalinity of the blood, and a consequent increase in the 
amount of uric acid, has been disproved. 

In order to test for uric acid in the blood, the following method 
may be employed : 100 to 300 c.c. of blood, obtained by means 
of cupping-glasses, are at once diluted with three or four times 
their volume of water and heated on a water-bath. As soon ^ as 
coagulation sets in, a few drops of a 0.3 to 0.5 per cent, solution 
of acetic acid are added until a feebly acid reaction is obtained. 



THE PROTEIDS OF THE BLOOD. 49 

After having been kept upon the boiling water-bath for from fifteen 
to twenty minutes longer, until the albumin has separated out and 
settled in brownish flakes, the mixture is filtered while hot, and 
the precipitate washed repeatedly with hot water. Filtrate and 
washings, which usually present a slightly yellow or brownish color, 
are again brought to the boiling-point after the addition of 0.3 to 
0.5 per cent, of acetic acid, decanted, filtered, and after the addition 
of a small amount of disodic phosphate further treated according 
to the Ludwig-Salkowski method (see Urine). The first filtrate is 
then treated with hydrochloric acid, evaporated to about 10 c.c, and 
allowed to stand for twenty-four hours, when the uric acid that has 
separated out is filtered off through asbestos or glass-wool. The 
filtrate may then be examined for xanthin-bases according to the 
same method. If no uric acid crystallizes out, as not infrequently 
occurs, the acid fluid is directly examined for uric acid by means 
of the murexid test (which see). If upon the addition of ammonia 
no distinct red color develops, the residue, after thorough desicca- 
tion, is dissolved in water, when a reddish color may be regarded 
as indicating the presence of uric acid, while a yellow or brown 
color is referable to xanthin-bases. Hopkins' method may also be 
used. 

Garrod's Test. — This test may be advantageously employed if it 
is desired merely to determine whether or not large amounts of uric 
acid are present in the blood. A few cubic centimeters of blood- 
serum (5-10) or of serous fluid, obtained by means of a blister, are 
placed in a watch-crystal and treated with from six to ten drops 
of a 30 per cent, solution of acetic acid. A linen thread is im- 
mersed in the fluid, which is then kept at a low temperature for 
from twelve to twenty-four hours. At the expiration of this time a 
few uric acid crystals will have separated out upon the thread, if the 
substance is present in large amounts. The true nature of these 
crystals may then be further determined by the microscope and the 
murexid test (see Uric Acid in the Urine). 

Literature.— Picard, Virchow's Archiv, vol. ii. p. 189. Garrod, Med.-Chir. 
Trans., 1854, p. 49. Salomon, Zeit. f. physiol. Chem., vol. ii. p. 65 ; and Charite Annalen, 
1880, vol. v. p. 137. Klemperer, Deutsch. med. Woch., 1895, No. 40. Weintrand, 
Ibid., V. B. p. 185. 

Xanthin-bases. — Xanthin-bases do not occur in normal blood or 
are present only in exceedingly small amounts. Under pathological 
conditions, however, they may be encountered in recognizable quan- 
tities, as in leukaemia, typhoid fever, lymphatic tuberculosis, emphy- 
sema, phthisis pulmonalis, pleurisy, and chronic nephritis. 

The method above indicated for the demonstration of uric acid 
in tiie blood should also be employed when it is found desirable to 
test for these bodies (see Urine). 

Literature.— A. Kossel, Zeit. f. physiol. Chem., 1882, vol. vii. p. 22. Scherer, Ver- 
nandl. d. physik. med. Ges. z. Wiirzburg, 1852, vol. ii. p. 325. 
4 



50 THE BLOOD. 

Fat and Fatty Acids. 

Quite recently Engelhardt has pointed out that the amount of 
fat which is contained in normal human blood may be subject to 
considerable variations, and gives 0.194 per cent, as the average. 
The lowest figure which he obtained was 0.101 and the highest 
0.273 per cent. These figures differ very materially from those of 
other observers, who have found from 0.73 to 1.4 per cent., but it 
is quite likely that Engelhardt's method is responsible for these 
differences, and is probably more reliable (see below). Unfortunately 
only a few analyses of pathological material have been made with 
this method, and these have reference only to the blood of cachectic 
individuals. An increase in the amount of fat has here not been 
demonstrated, the results varying between 0.112 and 0.284 per cent., 
with 0.174 as an average. The cachexias in question were of 
tubercular and carcinomatous origin. With the older methods an 
increase in the amount of fat, aside from that observed after the 
ingestion of large amounts of fatty food, has been met with in 
cases of obesity, chronic alcoholism, in phosphorus-poisoning, in 
injuries affecting the long bones and the spinal cord, in various 
hepatic diseases, chronic nephritis, tuberculosis, malaria, cholera, 
during starvation, pregnancy, in infants at the breast, etc. The 
greatest increase, however, is observed in certain cases of severe 
diabetes, in which amounts varying between 1.276 and 18.12 per 
cent, have been encountered, and in which the fat may be visible 
with the naked eye (see below). 

In such cases fat emboli may be found post mortem, plugging 
the vessels of various organs, and notably the brain, the lungs, 
and the kidneys. This increase in the amount of fat constitutes 
the condition spoken of as lipcemia. The term lipacidcemia has been 
applied to the occurrence of volatile fatty acids in the blood, noted 
by v. Jaksch in various febrile diseases, leukaemia, and at times in 
diabetes, in which this condition is supposed to stand in a causative 
relation to the coma. /5-oxybutyric acid has been found post mor- 
tem in the blood in diabetes. 

To demonstrate the presence of fat in the blood, it is best to pre- 
pare cover-glass specimens, and to mount these in a drop of a 5 per 
cent, solution of osmic acid. The fat droplets are thus colored 
black, and appear about as large as the finest fat granules which are 
found in milk or butter. They may also be stained with Sudan III., 
and are thus colored red. In every case the necessary instruments 
and glasses should be carefully cleansed with ether, so as to avoid 
the accidental introduction of fat. 

As a quantitative estimation of the fat is not always possible, 
Zancly recommends the following simple procedure to demonstrate 
the presence of an excess of fat : A small drop of blood is received 



THE PROTEIDS OF THE BLOOD. 



51 



Fig. 13. 



upon a cover-glass, which is then adjusted over the depression of a 
cupped slide and ringed with vaselin. On standing, the serum 
separates out concentrically or excentrically from the small blood- 
clot, and normally or in the presence of no excess of fat appears 
perfectly clear. If, however, much fat is present, it becomes cloudy 
after several minutes or hours, and then appears bluish white, 
grayish white, or even milky white. To ascertain positively that 
the turbidity is due to fat, a microscopical examination of the hanging 
drop is made within a few hours following the preparation of the 
specimen, so as to exclude fibrin as the possible cause of such 
turbidity. 

Quantitative Estimation. — The apparatus which is best used is 
a modification of that of Nerking, as 
suggested by Engelhardt. 1 As seen 
from Fig. 13, it consists of the ether 
flask A, which is placed on a permanent 
water-bath, such as that of Miinke. 
a represents the escape tube for the 
ether vapor ; at b there is a closure by 
means of mercury, the upper escape 
tube c dipping into the mercury over 
the mouth of b. B is the cooler for 
the ether vapor ; C, the water conden- 
ser. The cooled ether falls through 
the cooler into d. This ends below 
with a funnel-shaped mouth, close to 
the bottom of the extraction flask E, 
with five apertures, and has a small 
open side tube, /, which counteracts 
any negative pressure that may occur 
above the liquid in the extraction flask. 
The fluid to be extracted extends to 
within 1—2 cm. from the aperture of 
the off-flow tube i. When the ether 
layer extends to the level with k the 
tube i acts as a siphon and draws off 
the fatty ether into A again by way 
of the tube I, which is likewise pro- 
vided with a mercury stop. 

The blood, about 10 c.c, is received 
in a graduate and weighed. It is 
washed into the extraction flask with 
about ten times its volume of 2 per 
cent, hydrochloric acid and boiled for three hours (with inverted 
condenser). On cooling, the material is extracted in the appa- 

1 The apparatus may be procured from Arno Haak, Jena. Price, 12 marks. 




Fat-extraction apparatus. 



52 THE BLOOD. 

ratus described for about forty-eight hours. At the expiration of 
this time the fatty ether in A is poured into a separating funnel 
together with the ethereal washings, which are used to remove all 
the material from the flask, the idea being to get rid of any water 
or bits of the bloody material that may by chance have been siphoned 
into A. The ether is then evaporated in an open glass dish. The 
residue is dissolved in absolute ether and filtered through a double 
folded filter (so as to absorb any traces of water remaining) into a 
beaker, when the ether is allowed to evaporate. The residue is 
placed in a drying-oven at 40° C. for one hour, and after remaining 
in the vacuum over sulphuric acid for twelve hours it is weighed. 

With this method lecithins, cholesterins, and fatty acids are ob- 
tained conjointly with the fat, which Engelhardt does not regard as 
objectionable, as they are present only in traces and may be regarded 
as physiologically equivalent to neutral fat. 

To test for fatty acids, 20 to 30 c.c. of blood, obtained by means 
of cupping-glasses, are treated with an equivalent weight of sodium 
sulphate and boiled. The filtrate is then evaporated to dryness and 
extracted with absolute alcohol. Upon evaporation of this solution 
fa«tty acid crystals will be obtained, which can readily be recognized 
with the microscope (see Feces). 

Literature. — M. Bonninger, "On the Methods for the Estimation of Fat in the 
Blood, and the Amount of Fat in Human Blood," Zeit. f. klin. Med., vol. xlii. Parts 1 
and 2. T. B. Futcher, " Lipsemia in Diabetes Mellitus," Jour. Am. Med. Assoc., 1899, 
p. 1006. S. Wat j off, " Ueber d. Fettgehalt d. Blutes b. Nierenkrankheiten," Deutsch. 
med. Woch., 1897, p. 559. v. Jaksch, "Lipacidsemie," Zeit. f. klin. Med., vol. xi. W. 
Ebstein, " Beitrag z. Lehrev. d. Lipsemie u. d. Fettembolie," etc., Virchow's Archiv, 
1899, vol. civ. p. 571. M. Engelhardt, Deutsch. Arch. f. klin. Med., 1901, vol. lxx. 
p. 182. Zandy, Ibid., vol. lxx. p. 301. 

Lactic Acid. 

There appears to be some doubt whether or not lactic acid nor- 
mally occurs in the bipod of man during life. In the blood of dogs, 
however, Gaglio could always demonstrate the presence of the acid 
during the process of digestion, after feeding with meat. The 
amount varied between 0.3 and 0.5 pro mille. During starvation 
smaller amounts were found, but it never disappeared altogether. 
In one instance Gaglio obtained 0.17 pro mille after fasting for 
forty-eight hours. Similar results were obtained by Irisawa, who 
noted, moreover, that the amount of lactic acid in the blood stood in 
direct relation to the degree of anaemia which was produced. 

In the human being Irisawa found lactic acid fairly constantly 
after death, the amount, determined as zinc lactate, varying between 
0.233 and 6.575 pro mille. These extensive variations he was unable 
to explain by the character of the disease causing the fatal termina- 
tion, and it is possible that the cause therefore lies in the fact that 
in some cases the blood was obtained shortly after death, while in 
others many hours had elapsed, as Irisawa himself suggests. 

The following method may be employed : 100 to 300 c.c. of blood 



THE PROTEIDS OF THE BLOOD. 53 

are extracted with three times its volume of alcohol, filtered, and 
the filtrate evaporated to a syrupy consistence. This is then made 
strongly alkaline with barium hydrate and shaken with large quan- 
tities of ether, in order to remove the fats which are present. The 
residue is acidified with phosphoric acid and again shaken with ether 
for twenty minutes at a time, until the process has been repeated 
five or six times, the lactic acid passing over into the ether. The 
ether is distilled off from the extract, the residue taken up with 
water, and the solution carefully evaporated in order to drive oif any 
ether still remaining, as well as the fatty acids. Carbonate of zinc 
is now added and the solution heated to 100° C. and filtered. The 
filtrate is evaporated on a water-bath until crystallization begins, 
when it is allowed to cool and treated with a few drops of absolute 
alcohol, in order to effect a complete separation of the lactate of zinc. 
The solution is allowed to stand exposed to the air until a constant 
weight is obtained.. 

Literature. — G. Gaglio, " Die Milchsaure d. Blutes," Du Bois Arcliiv, 1886, p. 400. 
T. Irisawa, " Ueber d. Milchsaure im Blut und Harn," Zeit. f. physiol. Chem., 
1892, vol. xvii. p. 349. 

Biliary Constituents. 

Biliary constituents — i. e., bile-pigment and biliary acids — are not 
encountered in the blood under normal conditions, but are found 
whenever they are present in the urine (which see). It is noteworthy, 
furthermore, that bilirubin may frequently be demonstrated in the 
blood when a urinary examination in this direction yields negative 
results. According to v. Jaksch, 1 moreover, bilirubin occurs in the 
blood in nearly every case in which urobilin exists in the urine, 
which suggests that bile-pigment circulating in the blood may possi- 
bly be transformed into urobilin in the kidneys. 

A cholcemia is encountered in the various pathologic conditions 
which are associated with a resorption of bile, as in obstructive 
jaundice, in association with an excessive elimination of bile into 
the intestinal canal, as well as with an increased destruction of red 
corpuscles. 

In order to test for biliary acids, the blood is first treated with 
alcohol, in order to remove the proteids. The biliary acids which 
are present in the filtrate are next transformed into their lead salts 
by means of lead acetate and ammonia, and thus precipitated. 
After washing with water the precipitate is boiled with alcohol and 
filtered. The lead salts are decomposed by means of sodium carbo- 
nate, the solution is again filtered, the filtrate evaporated to dryness, 
and the residue extracted with absolute alcohol. The alcohol is dis- 
tilled oif, when the biliary salts of sodium will crystallize out or 
remain behind as an amorphous mass, which may be tested directly 

1 v. Jaksch, Clinical Diagnosis, 4th ed., 1896, p. 97. 



54 THE BLOOD. 

according to Pettenkofer's method. To this end, some of the residue 
is dissolved in water and treated with two-thirds of its volume of 
concentrated sulphuric acid, care being taken that the temperature 
does not rise beyond 60° C. To this mixture a few drops of a 20 
per cent, solution of cane-sugar are added, when in the presence of 
biliary acids a beautiful violet color is obtained, which is referable 
to the action of furfurol, formed from the cane-sugar and the acid, 
upon the biliary acids. 

Bilirubin can be demonstrated in the blood most readily in the 
following manner : 10 to 15 c.c. of blood obtained by means of 
cupping-glasses, are allowed to coagulate, when the serum is removed 
by means of a pipette, filtered through asbestos, and coagulated in 
as thin a layer as possible at a temperature of 80° C. Under such 
conditions normal serum presents a light straw color, while in the 
presence of biliary coloring-matter a light greenish color is seen, 
which becomes grass green on standing. Should the serum contain 
haemoglobin, as in hsemoglobinsemia, a brownish color results. 

Acetone. 

Acetone has been found in the blood in considerable amounts 
under various pathological conditions, and especially in fevers. 

In order to demonstrate its presence, the blood is first extracted 
with ether and subsequently distilled, when the distillate is tested as 
indicated elsewhere (see Acetonuria). 

Dennige's test may also be employed, and has the advantage of 
greater simplicity : 3 c.c. of blood are treated with about 30 c.c. of 
Dennige's reagent and allowed to stand until the dark -brown precipi- 
tate has settled to the bottom. The supernatant fluid is filtered off 
and treated with a little more of the reagent, so as to insure complete 
precipitation. It is then acidified with sulphuric acid and heated as 
described. The formation of a white precipitate, which is soluble 
in an excess of hydrochloric acid, is referable to acetone or diacetic 
acid. 

Literature. — v. Jaksch, Acetonurie u. Diaceturie, Berlin, 1885. Eeale, Schmidt's 
Jahrbxich., 1892, p. 106 (Extract). 

MICROSCOPICAL EXAMINATION OF THE BLOOD. 

The Red Corpuscles. 

Variations in Size and Form. — The normal red blood-corpuscles 
are greenish-yellow, circular, biconcave disks, which in post-embryonic 
life are non-nucleated. Their diameter varies between 6 and 9 f- } 
with an average of 7.5 fi. The presence of larger or smaller cells 
is abnormal. Smaller cells are termed microcytes, and measure from 
3.5 to 6 fi ; larger cells are known as macrocytes or megalocytes, and 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 55 

usually have a diameter of from 9.5 to 12 fx ; still larger specimeus 
are spoken of as giant corpuscles (Hayem) ; l they may attain a 
diameter of 16 /i. The terms microcytosis or microcythoemia and 
macrocytosis or macrocythcemia are used to designate a predominance 
of the corresponding variety. 

As regards the origin of the macrocytes, there is evidence to show 
that they may result from the common normocytes in the circulating 
blood through increased imbibition of plasma, so that their occurrence 
from this point of view could be regarded as a degenerative phenom- 
enon. But, on the other hand, the presence of macrocytes may be 
interpreted as evidence of a regenerative process, bearing in mind 
that in the bone-marrow the size of the erythroblasts is larger than 
that of the common normocytes ; the macrocytes would thus repre- 
sent young normocytes which have prematurely found their way 
into the circulation. The microcytes probably result from the normo- 
cytes in the circulating blood through loss of plasma ; whether their 
presence may at any time be regarded as the expression of a regen- 
erative process seems questionable. Not infrequently microcytes 
are formed artificially during the preparation of the specimen. 

Microcytosis is, on the whole, of comparatively little clinical 
interest, and may be observed in any severe anaemia. Macrocytosis 
is more important. To a certain extent it is seen in severe forms 
of anaemia of whatever origin, but it is noteworthy that the presence 
of macrocytes in large numbers is essentially observed in pernicious 
anaemia. During the active period of the disease the macrocytes 
may here represent 70 per cent, of all red cells (Lazarus). The 
condition, however, is not constant. 

Going hand in hand with pathological variations in the size of 
the red corpuscles, there are variations in form which may affect not 
only the microcytes and macrocytes, but also the corpuscles of normal 
size. Cells may thus be seen which resemble a flask, a kidney, a 
biscuit, a boat, a balloon, a dumb-bell, or an anvil, while others are 
altogether irregular in appearance (Plate II., Fig. 2). Especially 
interesting is the fact that such abnormally formed cells, which are 
generally spoken of as poikilocytes, may manifest a certain degree of 
motility, so that they have at times been mistaken for microparasites. 
This is seen especially in marked cases of pernicious anaemia, and is 
most noticeable in the smaller forms. In pernicious anaemia poihilo- 
cytosis is most pronounced, and at one time it was thought that the 
condition was characteristic of the disease. It has been shown, how- 
ever, that it occurs in other anaemias as well, though its occurrence 
is probably always evidence of a specially severe form. In chlorosis 
it is usually only seen in the most severe cases, and particularly in 
those manifesting a tendency to thrombosis and embolism. 

In this connection a special deviation from the normal form of the 

1 Hayem. Le Sang, Paris, 1891. 



56 THE BLOOD. 

red corpuscles also requires consideration, viz., the prevalence of 
oval cells. These are notably observed in pernicious anaemia and 
seem to be of distinct diagnostic importance. They are found not 
only during the active periods of the disease, but frequently also in 
the interval between exacerbations. 

Poikilocytosis is a degenerative phenomenon, and it is essential 
not to mistake for the pathological variety certain abnormalities in 
form which may be seen in any normal preparation. Such deviations 
from the circular form are the result of mechanical injury, mutual 
compression, etc., and can readily be distinguished with a little 
practice. 

In wet preparations red cells will be seen near the margin of the 
drop where evaporation is actively going on, which present little 
knobs or spicules on their surface and along the periphery. Such 
cells are spoken of as crenated cells. The phenomenon in itself is 
normal, but it is noteworthy that crenation may at times be observed 
in the centre of a carefully prepared specimen after a few seconds, 
while as a rule from fifteen to thirty minutes elapse before the process 
of crenation begins to attack cells iu this location. The significance 
of this early crenation is not known. This is also true of delayed 
money-roll formation, which is at times observed in various hepatic 
diseases, in pneumonia, nephritis, ate, whereas normally the red 
corpuscles tend to agglutinate in this form immediately unless special 
pains have been taken to secure the separation of the individual 
cells (Plate II., Fig. 1). 

Variations in the Color of the Red Corpuscles.— The degree 
of coloring of the red corpuscles depends upon the amount of 
haemoglobin. Owing to the biconcavity of the cells the centre in 
well-mounted specimens is always paler than the periphery, and 
any deficiency in the amount of coloring-matter is here at once 
apparent. With a moderate grade of anaemia the cell as a whole 
looks paler, and the pale central area is increased in size. With a 
further increase in the loss of coloring-matter the central area is 
absolutely colorless and encroaches upon the peripheral colored zone 
more and more until finally the so-called pessary forms result, in 
which only a narrow rim of haemoglobin remains. These changes 
can be made out in wet preparations, but are especially well seen in 
stained specimens. The central pale area is, however, visible only 
in well-preserved cells ; in every spread many corpuscles will be 
seen which appear merely as flattened disks, and are uniformly 
colored throughout ; the biconcave structure has here been lost as 
the result of mechanical injury. 

The color of the normal red cells in wet specimens is a pale 
greenish yellow. In malaria curiously discolored corpuscles are 
frequently seen, which present a bronzed appearance ; their presence 
should always excite suspicion. The meaning of the discoloration is 



PLATE I 



t, 



\ 4 













flic 



^9 ' 






8> 




LS. 



The Elements of Normal Blood. 



a, red cells in rouleaux ; <5, crenated red cells ; c, finely granular (neutrophilic) leucocytes ; a, 
coarsely granular (eosinophilic) leucocytes ; e, small, and /". large mononuclear leucocytes ; g, plaques. 



FIG. 2. 



^ 



/" N 




^5 


r . * 1 






r% ' : 








, .:"-■■■ 




Poikiloeyl 


:osis. 









-O 



^5 



Unstained specimen taken from a case of pernicious anaemia. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 57 

not known, but in all probability it is evidence of a degenerative 
process. 

The Color Index. — The term color index is used to designate the 
relative amount of haemoglobin which is contained in each corpuscle. 
It is determined by dividing the percentage of blood coloring-matter 
by the percentage of red cells as compared with the recognized 
normal, viz., 5,000,000. 

Example. — The percentage of haemoglobin is 50, the red count 
per cbmm. is 2,000,000, viz., 40 per cent, of the recognized normal, 
5,000,000. The color index is then 50 divided by 40 — I e., 1.25. 

Under normal conditions the color index is about 1, but may vary 
from 0.95 to 1.17 ; it is slightly higher in men than in women. In 
the secondary anaemias, in which the decrease in the amount of 
haemoglobin is proportionate to the diminution of the red corpuscles, 
the color index is approximately normal. But in the majority of 
cases the diminution of the haemoglobin somewhat exceeds that of 
the red cells, so that lower values are commonly met with. In 
pernicious anaemia, on the other hand, w T here the corpuscular decrease 
usually exceeds the diminution of the haemoglobin, a high color index 
is the rule. There may be periods in the course of the disease, how- 
ever, in which a normal index and even subnormal values are found. 
In the chronic cases lower figures are more commonly obtained than 
in the acute cases. In the series of 22 cases collected by Strauss and 
Rohnstein the value of the color index varied between 0.5 and 1.95. 
In 8 cases of the series variations from 1.13 to 1.95 were observed, 
and in 6 lower values than 1 were noted, viz., 0.5-0.9. Cases in 
which the color index falls as low as 0.5 are rare in pernicious 
anaemia. In the one instance in the series in which this was found, 
the haemoglobin was only 10 per cent., while the red cells numbered 
1,048,000 ; there was a high grade of poikilocytosis and all transitions 
between the smallest microcytes and the largest types of macrocytes. 

In the secondary anaemia of carcinomatosis the color index rarely 
exceeds 1. In Strauss and Rohnstein's series 1 of 35 cases the highest 
value was 1.1 (in one case only) ; in the rest it varied between 0.53 
and 0.96. 

In chlorosis, in which the degree of corpuscular diminution usually 
exceeds that of the haemoglobin, the color index is markedly lower ; 
in especially severe cases it may fall to 0.3 and even lower. But it 
is not admissible to make the diagnosis of chlorosis on this basis 
only, as it is fairly common to meet with a markedly lowered color 
index in some secondary anaemias also, and especially in the form 
which is referable to carcinoma. In splenic anaemia likewise the 
degree of oligochromaemia may far exceed the degree of oligocy- 
themia. 



1 Strauss u. Rohnstein, Die Blutzusanimensetzung b. d. verschiedenen Anaemien, 
Berlin, 1891. 



58 THE BLOOD. 

Variations in Number. — The number of red corpuscles in the 
blood of healthy adults is fairly constant. In man 5,000,000 may 
be considered a fair average, and in women 4,500,000. Higher 
values are not uncommon, but rarely exceed 6,000,000 in perfectly 
normal individuals. 

The highest figures are normally obtained at birth. In a study 
of seventeen infants Hayem found 6,260,000 as the highest and 
4,340,000 as the lowest count. Soon after birth the number dimin- 
ishes, but later it rises again, until the normal adult values become 
established about the time of puberty. 

A somewhat higher average is found among people living at a 
considerable elevation above the sea level, and it is interesting to 
note that an increase in the number occurs whenever a change in 
the habitation is made from a lower to a higher level. This increase 
is frequently quite marked, as is apparent from the following table, 
which is taken from Ehrlich : l 

Altitude Increase of 

561 metres 800,000 

700 " 1,000,000 

1800 " 2,000,000 

4392 " 3,000,000 

A corresponding diminution occurs when a change is made from a 
higher to a lower level. 

In this connection GauleV observations are especially interesting. 
On the occasion of a balloon ascension to a height of from 4200 to 
4700 metres he counted 7,040,000, 8,800,000, and 7,480,000, 
respectively, in the three participants of the journey. The haemo- 
globin was at the same time diminished, and he accordingly con- 
cluded that the increase during the ascent was due to an increased 
production of red cells ; the probable nature of this conclusion was 
further strengthened by the fact that numerous normoblasts were 
found in the blood, many undergoing division. Jolly and Bensaude 3 
on similar expeditions were unable, however, to demonstrate the 
presence of nucleated red cells. According to Weinzirl, 4 the 
increased counts due to high altitude are temporary and in part at 
least referable to cold. He showed that in rabbits a certain increase 
in the number of red cells occurs when they are removed from warm 
to cold quarters, and that their subsequent removal to a higher alti- 
tude does not lead to a further increase. 

Clinically we distinguish between relative polycythcemia in which 
the condition is due to a diminution in the quantity of the plasma, 

1 P. Ehrlich u. A. Lazarus, " Die Anseinie," Nothnagel's specielle Path. u. Therap., 
Vol. viii. part 1. 

2 Gaule, Compt. rend., vol. cxxxiii. p. 903. 

3 Bensaude, Compt. rend. Soc. biol., vol. liii. p. 1084. 

4 Weinzirl, Am. Jour. Med. Sci., 1903, vol. cxxvi. p. 299. 



3IICR0SC0PICAL EXAMINATION OF THE BLOOD. 59 

and true polycythemia in which there is an actual increase in the 
number of the red corpuscles. Relative polycythemia is much the 
more common. It may be referable to loss of liquid, either by 
sweating, diarrhoea (by far the most common), or increased diuresis. 
In another group of cases there is loss of liquid by secretion or 
transudation, as in narrowing of the pylorus with dilatation of the 
stomach, and in the constant loss of liquid from the blood in 
recurring ascites. In some of these cases the polycythemia is of 
high grade, and may persist for years. The polycythemia which is 
noted in poisoning by phosphorus and carbon monoxide, during and 
immediately after the administration of ether, following cold baths 
and severe muscular exercise, probably also belongs to this order and 
is no doubt referable to vasomotor disturbances. Of similar origin 
no doubt is the polycythemia which is noted in disease of the adrenal 
glands, where counts of from 6,000,000 to 7,000,000 have been 
repeatedly noted ; and the same is probably true of diabetes, in 
which polycythemia may be observed both while fasting and while 
much fluid is being ingested. 

True polycythemia is met with in diseases in which there is difficulty 
in proper aeration of the blood, as in heart disease, 1 and in a peculiar 
type of chronic cyanosis which has recently been described by Osier 2 
as a new clinical entity. In acquired heart disease with continued 
inadequacy of the circulation of slight degree a moderate grade of 
polycythemia is very common ; in the congenital form the figures 
often reach 8,000,000 to 9,000,000. The highest values are seen 
in Osier's disease. In the nine cases which have been thus far 
reported the highest count was 12,000,000 ; in eight of the cases it 
was above 9,000,000, and in the ninth it was 8,250,000. The 
usual range of hemoglobin at the same time was from 120 to 150 ; 
the specific gravity varied between 1.067 and 1.083, and the leuco- 
cyte count between 4000 and 20,000 ; as a rule it was below 10,000. 

While there can thus be no doubt that a true polycythemia does 
occur, it has been conclusively demonstrated that such a condition 
does not exist in what is generally termed true plethora, and that 
the various symptoms of plethora formerly attributed to a general 
increase in the amount of blood are more likely referable to vaso- 
motor disturbances. 

Oligocythcemia, viz., a diminished number of red cells, is much 
more common than polycythemia. It may be temporary or perma- 
nent, and is seen in all forms of anemia of whatever origin. It is 
most marked in pernicious anemia. The exact figure will here, of 
course, depend upon the stage of the disease and the individual case. 
A decrease to one-half of the normal number may be seen in com- 

1 Stengel, Proc. Path. Soc. Phila., 1899, Oertel, Deutsch. Arch., vol. 1. p. 293. 
2 W. Osier, "Chronic Cyanosis with Polycythemia," Am. Jour. Med. Sci., 1903, vol. 
cxxvi. p. 187. 



60 THE BLOOD. 

paratively mild cases ; a million red cells is a common count. The 
number may fall to 500,000 and even lower. In one case reported 
by Quincke 1 a count of 143,000 was observed, and it is interesting 
to note that seventy-four days later the same patient had 1,234,000 
per cbmm. Osier 2 reports a case in which shortly before death the 
red cells fell below 100,000. This is the lowest count that has been 
recorded. In the stage of amelioration they may rise to 4,000,000 
and even higher. In the series collected by Strauss and Rohnstein 3 
1,240,000 was the average at the time when the patient first came 
under observation, and in Cabot's series of one hundred and ten 
cases the average number is almost identical — 1,200,000. 

In chlorosis, contrary to what is found in pernicious anaemia, the 
red cells are usually not much diminished. In Cabot's 4 series of 
seventy-seven cases the average count was 4,050,000. At times, 
however, cases are met with in which the diminution of the red cells 
almost keeps step with the diminution in the amount of haemoglobin. 
v. Limbeck cites three cases with 1,750,000, 1,850,000, and 
1,930,000, respectively ; and Hay em mentions an instance in which 
only 937,360 cells were counted. Such cases are exceptional. 

As in chlorosis so also in splenic anaemia, the corpuscular anaemia 
is of very moderate grade, even though the diminution in the amount 
of haemoglobin may be considerable. Of the forth-one cases collected 
by Osier, the average was 3,425,000 ; the lowest count was 2,187,000 
and the highest 5,200,000. 

In leukaemia the red cells are usually not diminished to a very 
great extent ; and the oligocythemia is generally more marked in 
the lymphatic than in the myelogenous variety ; the average figures 
in Cabot's series are 2,730,000 and 3,120,000, respectively. Counts 
of 1,000,000 or thereabout may, however, be met with. 

In pseudoleukaemia the red cells may be only moderately dimin- 
ished, viz., between 3,000,000 and 4,000,000, but in some cases the 
corpuscular destruction is quite active, and in the last stages of the 
disease values may be found which are not much above 1,000,000 
or 1,500,000. 

An extreme and rapidly progressive anaemia is frequently noted in 
acute streptococcus infections. Grawitz 5 states that according to 
Rocher's investigations it is probable that the diminution of the red 
cells in septicaemia is greater than in any other infectious disease and 
appears in a shorter time. Cases may indeed be encountered in 
which the question of pernicious anaemia may enter into the diag- 
nosis, as occurred in two cases of gonorrhoeal endocarditis which 
were observed by Osier. 

1 Quincke, Centralbl. f. d. med. Wiss., 1877, No. 47 ; and Deutsch. Arch., 1877, vol. xx. 

2 Osier, Johns Hopkins Hosp. Bull., 1902, vol. xiii. p. 251. 

3 Strauss u. Eohnstein, loc. cit. 

4 Cabot, Clinical Examination of the Blood, Wra. Wood & Co. 
5 Grawitz, Klin, pathol. d. Blutes, Enslin, Berlin, 1902. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 61 

An extreme grade of corpuscular destruction is also noted in 
malaria. In acute cases the loss of red cells during the first twenty- 
four hours may reach 1,000,000, and in two days even 2,000,000. 
In neglected chronic cases the count usually varies between 3,000,000 
and 4,000,000 ; the oligocythemia may, however, be far more 
extensive, and Ewing 1 cites a case observed by Kelsch, with only 
583,000 red cells per cbmm. 

The anemia observed after typhoid fever is as a rule not very 
severe, but exceptional cases occur in which the loss of red corpus- 
cles is considerable. Osier cites an instance in which the number 
fell to 1,300,000. 

The post-rheumatic anaemia is usually not so pronounced. 

In acute endocarditis Stengel 2 has noted a rapid decrease of the 
red corpuscles, often to 50 and even 40 per cent. 

In pulmonary tuberculosis the number of the red corpuscles runs 
a course parallel to that of the haemoglobin (which see). Oligo- 
cythemia is really only seen during the third stage (2,000,000- 
2,500,000) ; while during the second stage, owing to an actual con- 
centration of the blood (Grawitz), normal figures are the rule. In 
the first stage a diminution of their number (3,800,000) is only seen 
in patients who have repeatedly suffered from tuberculous affections 
(scrofula) since childhood, and in whom the onset of the pulmonary 
disease has been gradual, while, on the other hand, normal values 
are found in individuals who appear to be in perfect physical health, 
who are well-nourished, with well-shaped chests, and without hered- 
itary predisposition (Appelbaum). 3 

In acute gastritis, and usually in chronic gastritis also, the number 
of the red corpuscles is not diminished, while in carcinoma a marked 
oligocythemia occurs at some time in the course of the disease. In 
the earlier stages, however, this is often but little marked, and at 
times even an apparent increase of the red cells is noted. Later a 
diminution is probably always found. In the severer forms of 
chronic gastritis a diminution is also fairly constant, but rarely so 
marked as in carcinoma, if we except those cases of gastric anadeny 
which present the clinical picture of a pernicious anemia. In the 
differential diagnosis between carcinoma of the stomach and perni- 
cious anemia a count below 1,000,000 points to the latter disease. 
In ulcer of the stomach normal values are found unless hematemesis 
has recently occurred or unless the disease is associated with pro- 
found chlorosis. 

The count which is obtained in post-hemorrhagic cases will depend 
very largely upon the amount of blood lost and the time at which 
the examination is made. The lowest counts, according to Lyon, 4 

1 Ewing, On the Blood, Lea Bros. & Co., 1901. 

2 Stengel, loc. cit., p. 59. 

3 Appelbaum. Berl. klin. med. Woch., 1901, vol. xxxix. p. 7. 

4 Lyon, Virchow's Archiv, 1881, vol. xciv. 



62 THE BLOOD. 

Htihnerfauth 1 , and Siegel-Maydl, 2 are found between the second and 
the eleventh day. In RiederV cases the figures varied between 
1,300,000 and 3,335,000 ; in those of Strauss and Rohnstein, 4 
between 1,119,000 and 4,420,000. A sudden reduction in the 
number to 1,000,000 or less is usually followed by a fatal result. 

In the anemias of infancy and early childhood the oligocythemia 
is often very pronounced. In the infantile pseudoleukemia of 
v. Jaksch especially low values may be found associated with an 
increase of the leucocytes of such extent that the ratio between 
the two may be suggestive of true leukaemia ; there is, however, no 
myelernia, but an increase of the normal types. In infantile leu- 
kaemia of the lymphatic variety McCrae found 2,350,000 as the 
highest count. 

In the majority of cases of rickets there is no material diminution 
in the number of the red cells, while the haemoglobin may be much 
reduced, but in the severer forms with visceral complications there 
may be oligocythemia of extreme grade, v. Jaksch cites a case in 
which the red count fell from 1,600,000 to 750,000 within three 
months, and Luzet noted a drop to 500,000 within three weeks 
(Ewing). 

In congenital syphilis the oligocythemia is usually marked, 
excepting in very mild cases, and in the severer infections the blood 
picture may simulate that of pernicious anemia. 

Behavior toward Anilin Dyes. — Polychromatophilia (Polychro- 
masia). — The normal living red cell possesses no affinity for dyes ; it 
is achromatophilic. The normal fixed cell of the circulating blood, 
on the other hand, has a marked affinity for acid dyes, such as eosin, 
orange-G, acid fuchsin, etc. ; it is accordingly said to be oxyphilic, 
and as it takes up only one color from a mixture of different dyes it 
is termed monochromatophilic. Under various pathological condi- 
tions which are associated with a marked grade of anemia cells are 
met with which are polychromatophilic. Such cells manifest an 
affinity not only for acid dyes, but simultaneously also for basic 
dyes, so that with a mixture of hematoxylin and eosin, or eosin and 
methylene-blue, for example, the red cells are not stained in the 
usual tint of the hemoglobin, but present a mixed color in which 
the tint of the basic dye is more or less apparent. With hema- 
toxylin and eosin, for example, some cells will be colored a bluish 
red, others a reddish blue, still others violet, and again others a pure 
blue (Plate X.). 

As regards the significance of the polychromasia, Ehrlich main- 
tained that the condition was evidence of a degenerative process — 
of a coagulation-necrosis of the discoplasm — during which this takes 

1 Hiihnerfauth, Virchow's Archiv, vol. lxxvi. 

2 Siegel-Maydl, Wien. med. Jahrbuch., 1884. 

3 Rieder, Beit. z. Kenntniss d. Leucocytose, Leipzig, 1892. 

4 Strauss u. Rotmstem, loc. cit. 



PLATE III 



.m. 






c 







• 



/ 



"-if: 






* 



^y 












• 



L.S. 



a, a group of red cells undergoing granular degeneration ; <5, red cells showing Cabot's 
ring bodies ; c, normoblasts with nuclei undergoing karyolysis, the bodies of the cells show 
granular degeneration ; d, normoblast with pvknotic nucleus; y, red cell, suggesting loss of 
nucleus b}' extrusion ; g, red cell undergoing mitosis ; h, megaloblasts with polychromasia of 
protoplasm ; z, gigantoblast ; k, nucleated red cells with wheel-shaped nuclei undergoing 
cytol3'sis ; /, a group of plaques. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 63 

up albumiDs from the blood-plasma, while at the same time it loses 
the power of holding its haemoglobin. As a consequence the oxy- 
philia diminishes, while owing to the absorption of albumins a more 
or less well-marked basophilia develops. As a matter of fact poly- 
chromatophilia is often seen in cells which are manifestly degener- 
ating, and in myelogenous leukaemia especially one frequently meets 
with nucleated red corpuscles which are markedly polychromatic 
and in which the protoplasm is evidently undergoing destruction, 
often appearing merely as a little hood attached to one side of the 
nucleus (see Plate III.). Ehrlich accordingly speaks of an ancemic 
or polychromatophilic degeneration of the blood. But, on the other 
hand, there is evidence to show that polychromasia may be the ex- 
pression of a regenerative process, and we find as a matter of fact that 
the erythroblasts of the normal bone-marrow are for the most part 
polychromatophilic, and the more markedly so the younger they are. 
Megaloblasts are probably always polychromatophilic (Plate III.). 
Welker has shown that basophilic red cells are normally found in 
pigeons, mice, guinea-pigs, cats, and dogs, while they are absent in the 
horse and the ox. I have also found them in the squirrel. In those 
animals, moreover, in which the red cells of the circulating blood are 
normally nucleated a certain grade of polychromasia, according to 
my experience, appears to be the rule in all the younger cells ; the 
pure haemoglobin tint is only found in the mature cell and in the 
oldest forms which are undergoing cytolysis. 

Of late, Ehrlich has admitted the existence of a physiological 
polychromasia, but he still maintains that it may also occur as the 
expression of a degenerative process. 

Literature. — Ehrlich, Charite Annalen, vol. x, p. 136. Engel, Deutsch. med. 
Woch., 1899, p. 209. Gabritschewsky, Arch. f. exp. Path., vol. xxviii. p. 83; Zeit. 
f. klin. Med., vol. xxvii. p. 492. Askanazy, Ibid., vol. xxi. p. 415. Maragliano and 
Castellino, Ibid., vol. xxi. p. 415. 

Diabetic Chromatophilia. — Bremer has pointed out that a distinct 
difference exists in the affinity of diabetic blood for certain anilin 
dyes, as compared with non-diabetic blood. For, whereas non- 
diabetic blood is readily stained with Congo-red, methyl-blue, eosin, 
etc., diabetic blood is distinctly refractory, while such dyes as 
Biebrich-scarlet, which readily stain the diabetic blood, do not color 
non-diabetic blood. Upon this peculiarity in the. behavior of the 
red corpuscles Bremer's diabetic blood test is based. 

Method. — A drop of blood of moderate size is mounted on a 
slide and spread out in a wave-like manner, using the edge of a 
second slide for this purpose. A number of such preparations are 
made, as also an equal number with normal blood for control. 
These are then placed on the tray of a drying-oven at a distance 
of 12 cm. from the bottom. The bulb of the thermometer is fixed 



64 THE BLOOD. 

at the same level. The temperature is then rapidly raised to about 
130° C, when the flame is removed. Care should be taken that 
the temperature thereafter does not exceed 140° C; the optimum 
lies at about 135° C. The apparatus is then allowed to cool until 
the preparations can be conveniently handled, when a specimen of 
the diabetic blood is placed back to back with a control-specimen, 
and both are immersed in the staining fluid. A 1 per cent, aqueous 
solution of Congo-red, which should always be made up freshly 
when required, is advantageously employed. After exposure for 
from one and a half to two minutes the specimens are rinsed in 
water and dried with filter-paper. It Avill then be seen that the 
non-diabetic blood is stained the color of Congo-red, while the 
diabetic blood is either not stained at all or presents merely an 
orange color. 

Other stains may also be employed, such as a 1 per cent, aqueous 
solution of methyl-blue or Biebrich-scarlet, or Ehrlich's tri-acid 
stain, the eosinate of methylene-blue, and others. When using 
methyl-blue analogous results are obtained as with Congo-red. 
With Biebrich-scarlet, on the other hand, the diabetic blood takes 
up the color, while the non-diabetic specimen proves refractory. 
If Ehrlich's stain is employed, an exposure to the stain for from 
two to five minutes is necessary ; the diabetic specimen is stained 
orange, the non-diabetic blood violet. 

Very satisfactory results are obtained also with the following 
method : the preparations are first stained for from one and a half to 
two minutes in a 1 per cent, aqueous solution of methyl-green. Upon 
washing, it will be seen that both specimens are colored green, but 
the diabetic blood more markedly so than the other. Both are then 
immersed for from eight to ten seconds in a 0.12 per cent, aqueous 
solution of eosin, when the diabetic blood remains green, while the 
non-diabetic specimen is colored eosin. Analogous results are 
obtained with methylene-blue and eosin. 

Success in these examinations depends essentially upon the proper 
degree of temperature during the process of fixation. But care 
should also be had not to leave the specimens in the staining solu- 
tion longer than indicated, and to rinse quickly in water and dry. 

Regarding the nature of the substance in diabetic blood which is 
responsible for this peculiar behavior, little is known, but it appears 
certain that the reaction is not dependent upon the presence of glu- 
cose nor upon the degree of alkalinity of the blood, as suggested 
by Lepine and Lyonnet. Bremer's claim that the reaction is pathog- 
nomonic of diabetes and glucosuria, and may even yield positive 
results in the pre-diabetic stage of the disease, and when the sugar 
has temporarily disappeared from the urine, has been confirmed in 
all essential points, both in this country and abroad. A few inter- 
esting exceptions, however, have been noted. In animals, for 






MICROSCOPICAL EXAMINATION OF THE BLOOD. 65 

example, in which glucosuria has been artificially produced by 
means of phlorhizin, the reaction does not occur, whereas in phloro- 
glucin-diabetes positive results are obtained. In Bremer's entire 
series of diabetic cases a negative result was obtained but once, and 
in this instance he believes that the diabetes was of the renal type, 
and analogous to the phlorhizin-diabetes of animals. He suggests 
that it may thus be possible to differentiate this form from the 
hsematogenic variety, using the latter term in its widest sense. 
Lepine and Lyonnet report a positive result in one case of leukae- 
mia, but Bremer believes this to have been due to faulty technique. 
Hartwig finds that Bremer's reaction is constant in diabetes, but that 
it may also occur at times in other conditions. 

Literature. — L. Bremer, ''An Improved Method of Diagnosticating Diabetes 
from a Drop of Blood," N. Y. Med. Jour., 1896 ; Centralbl. f. inn. Med., 1897, p. 521. 
Le Goff, React, chrom. du sang diabet., Paris, 1897. Lepine and Lyonnet, Lyon med., 
vol. lxxxii. p. 187. Hartwig, Deutsch. Arch. f. klin. Med., vol. lxii. p. 287. 

Granular Degeneration of the Red Cells. — Under pathological 
conditions red cells may be met with which contain basophilic 
granules. These are readily stained with methylene-blue, methylene- 
azure, thionin, etc. Methyl-green, however, which is a specific 
nuclear dye, does not stain the granules. Their size, form, and 
number are variable. While the majority are round, others are rod- 
or biscuit-shaped. The largest granules are found in pernicious 
ansemia and in cases of lead-poisoning w T ith intestinal manifes- 
tations. They are then quite readily seen and attract attention at 
once (Plate III.). In most other diseases in which they occur they 
are much smaller, and on superficial examination they may indeed 
be overlooked ; some cells at first sight merely look a little off-color, 
and it is seen only on very careful examination that the apparent 
polychromasia is in reality due to the presence of large numbers of 
minute dots. This finest variety is in my experience most com- 
monly seen in obscure cases of lead-poisoning, or in such cases which 
do not present intestinal symptoms. In other conditions the size is 
about that of a neutrophilic granule. Very often, in specially 
anaemic cells, the granules are arranged in the peripheral portion of 
the cell. Their number is exceedingly variable ; generally speak- 
ing, it depends upon their size ; when they are especially large they 
are relatively little numerous. 

The granules may occur in cells of normal size or color, in poiki- 
locytes, and in nucleated red cells, both of the normoblastic and the 
megaloblastic type, especially the former. Not infrequently they 
are seen in cells which are markedly polychromatic, but, like 
Grawitz, I do not believe that granular degeneration represents a 
later phase of polychromasia. 

In disease they are most constant and numerous in pernicious 
ansemia, in lead-poisoning, and in malaria ; they are less constant 
5 



66 THE BLOOD. 

and less numerous in the leukaemias, in pseudoleukemia, in the 
cachexias referable to septic infection, syphilis, carcinomatosis, and 
in the final stages of tuberculosis. In chlorosis and in the anaemia 
of chronic nephritis they are absent ; in a case of v. Jaksch's 
anaemia, in which nucleated red cells were quite numerous, I 
obtained negative results. 

In pernicious anaemia granule cells are frequently found in the 
interval and at a time when the blood picture is otherwise practically 
normal. I have seen them most numerous in a case in which blood 
crises occurred from time to time (see page ) ; almost every nor- 
moblast contained granules ; non-nucleated granule cells were, how- 
ever, at the same time present in large numbers. 

In lead-poisoning granule cells are practically found without 
exception, and may be encountered at a time when no clinical symp- 
toms are manifest. The amount of lead which is necessary to call 
forth their appearance is quite small, and it is a common experience 
to meet with a small number after the administration of lead in 
medicinal doses. I have found them after the ingestion of only 0.5 
gramme given in divided doses in the course of forty-eight hours. 
In cases of lead-poisoning they persist for a long time after exposure 
has ceased. In one case of double wrist- and ankle-drop I could 
still demonstrate granule cells after five months. 

In malaria granule cells are very common. Plehn found them 
in Europeans after a short sojourn in the tropics, and looked upon 
the granules as spores of the malarial parasite. 

In septic cases and in the cachexia of carcinomatosis they are not 
numerous ; in a case of cancer of the stomach with only 27 per cent, of 
haemoglobin, which I recently observed, I found no granule cells. 

In the early stages of phthisis granular degeneration is not seen, 
but it may occur later, when a general septicaemia has supervened. 

As regards the significance of the granules, Engel, Ehrlich, and 
others have suggested that they are most likely products of kary- 
olysis. But Grawitz, Stengel, Pepper, and White maintain, and I 
think rightly so, that they are not of nuclear origin. They may be 
found at a time when not a single nucleated red cell is demonstrable 
in the blood ; nucleated red cells may be seen in which no sign of 
karyolysis is manifest, while the body of the cells is studded with 
granules ; unlike the nuclei of the erythroblasts, the granules have no 
affinity for methyl-green, which is a specific nuclear dye ; they may be 
found in nucleated cells which are undergoing karyokinetic division. 

It is interesting to note that granule cells may not be found in 
the bone-marrow even when they are numerous in the circulating 
blood ; when they do occur, they are not more numerous than in the 
peripheral vessels. Grawitz hence regards their presence as an 
indication of a degenerative change in the haemoglobin, and speaks 
of the phenomenon as " granular degeneration," Schmauch, on the 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 67 

other hand, has observed similar appearances in the blood of healthy 
cats, and Engel has described the occurrence of granule cells in the 
blood of early cat embryos. I have found granule cells in the 
squirrel and in a porcupine suffering from filariasis. It is stated by 
all observers that they do not occur in the blood of man when in 
perfect health. My own experience has practically been the same, 
but I have found small numbers in two individuals who at the time 
considered themselves perfectly well. One of these at the same 
time had 6.5 per cent, of eosinophiles and had recently passed a 
round worm. 

In white mice Grawitz was able to produce a granular degenera- 
tion by prolonged exposure of the animal to a temperature of from 
37° to 40° C. He suggests that the analogous results which were 
obtained by Plehn in the case of Europeans after a brief sojourn in 
the tropics may possibly be referable to the high temperature. 

Literature. — E. Grawitz, " Ueber Kornige Degeneration d. rothen Blutzellen," 
Deutsch. med. Woch., 1899, No. 36, p. 585 ; " Klinische Bedeutung u. experiment. Er- 
zeugung korniger Degenerationen," etc., Berlin, klin. Woch., 1900, p. 181 ; "Granular 
Degeneration of the Erythrocytes," etc., Am. Jour. Med. Sci., 1900, vol. cxx. p. 277. 
Bloch, Deutsch. med. Woch., 1899, V. B. p. 279. Litten, Ibid., No. 44. Behrendt, 
Ibid., No. 44. White and Pepper, " Granular Degeneration of the Erythrocyte," Am. 
Jour. Med. Sci., 1901, vol. cxxii. p. 266. C. E. Simon, International Clinics, 1902, vol. 
i. p. 69. Stengel, White, and Pepper, Am. Jour. Med. Sci., 1902, vol. cxxiii. p. 873. 

Cabot's Ring Bodies, — Cabot has recently drawn attention to the 
occasional occurrence in red cells of curious ring bodies which are 
usually stained red with Wright's modification of Leishman's stain, 
but which may also take on a blue color. He found such rings in 
three cases of pernicious anaemia, in three of lead-poisoning, and in 
one case of lymphatic leukaemia. I have been able to demonstrate 
the same structures with the eosinate of methylene-blue, and could 
verify Cabot's observation that they occur in granule cells, but may 
also be found in apparently normal red corpuscles (Plate III.). No 
doubt they bear some relation to the nucleoids. 

Literature. — Cabot, Jour. Med. Eesearch, 1903, vol. ix. 

Ehrlich's Haemoglobinaemic Innenkbrper. — These structures may be 
encountered in red cells in conditions associated with extensive 
hsemocytolysis the result of specific blood poisons. The individual 
body is round and characterized by its affinity for acid dyes. 

Nucleated Red Corpuscles. — Nucleated red corpuscles are not 
found in the circulating blood of healthy individuals, excepting at 
birth and during the first days of life, when it is not unusual to 
meet with an occasional cell of this type. In the bone-marrow, 
however, they are always found. It is here possible to distinguish 
two types, viz., the normoblast and the megaloblast. The latter is 
ontogenetically the older and gives rise to the normoblast through a 
process of homoplastic differentiation by cell division ; it thus bears 
the same relation to the normoblast which exists between the large 



68 THE BLOOD. 

lymphocyte and the small lymphocyte, and the amblychromatic 
myelocyte and the trachychromatic myelocyte (see page ). The 
megaloblast itself results from the large lymphocyte through direct 
heteroblastic transformation and ages into the niacrocyte, while the 
normoblast similarly develops into the normocyte (Pappenheim). 
In the bone-marrow of adults megaloblasts are only found in small 
numbers ; the great majority of nucleated red cells are of the nor- 
moblastic type. 

The normoblasts (Plate III.), like the normal red cells of the 
circulating blood, have a diameter which varies from 6 to 9 p.. 
The nucleus in the youngest cells occupies a central position, and is 
larger and relatively poorer in chromatin than in the older cells, 
where it is frequently located excentrically. The size varies between 
2 and 4 fi. In the younger cells the chromatin is quite commonly 
arranged in a stellate manner, while later the nuclear juice manifestly 
diminishes in amount, and in the oldest cells the nucleus is accord- 
ingly much smaller and markedly pyknotic ; but in all cells it stains 
quite deeply. 

The protoplasm in the majority of the normoblasts of the bone- 
marrow is polychromatophilic, while in most of the cells which are 
found in the peripheral blood in disease the oxyphilic tendency 
prevails. But at times here also the normoblasts are polychromato- 
philic. With the triacid stain a few cells of this type, in the bone- 
marrow, take on the fuchsin stain (Engel's fuchsinophilic cells). 

In cases of pernicious anaemia, in myelogenous leukaemia, and in 
lymphatic leukaemia (notably the acute form) normoblasts are at 
times seen which are undergoing mitosis (Plate III.). 1 Much more 
common, however, are cells in which the nucleus is more or less 
lobed, and presents appearances which are commonly interpreted as 
evidence of beginning karyolysis (Plate III.). Free nuclei may like- 
wise be seen in the blood. 

In the majority of cases in which normoblasts are found in the 
blood these are well preserved, but in myelogenous leukaemia more 
especially it is very common to meet with cells in which the proto- 
plasm surrounding the nucleus is much diminished in amount and 
presents a ragged outline. These cells are manifestly degenerating, 
and in many specimens the protoplasm will be seen reduced to a 
little hood which is attached to one side of the nucleus (Plate III.). 
Such cells in my experience are always polychromatophilic and are 
apt to be mistaken by the beginner for poorly stained lymphocytes. 

The occurrence of normoblasts in the circulating blood is always 
evidence of stimulation of the bone-marrow, which may occur either 
indirectly, as the result of an " anaemic " condition of the blood, or 
directly, as in disease of the bone-marrow per se (Grawitz). We 
may accordingly meet with normoblasts in almost any form of 

1 G. Dock, " Mitosis in Circulating Blood," Trans. Assoc. Am. Phys., 1^>02, p. 484. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 69 

anaemia, be this the result of traumatism (post-hernorrhagic), of 
inanition, or of organic disease. In the acute forms of anaemia they 
are apt to be most numerous, but even in the more chronic cases 
and in cachectic conditions specimens of blood may be obtained in 
which one or more normoblasts are seen in every field. In the 
secondary anaemias, however, it is usual to meet with only a few 
nucleated cells. 

At times there appears to occur a sudden invasion of the cir- 
culating blood by red cells, many of which are nucleated ; this 
phenomenon v. Noorden terms a blood crisis, and it is noteworthy 
that the invasion of the red cells may be preceded and accompanied 
by a very extensive increase of the leucocytes. Ehrlich cites a case 
of hemorrhagic anaemia, reported by v. Noorden, in which at the 
time of such a blood crisis the normoblasts were so numerous, while 
hyperleucocytosis of high grade existed at the same time, that the 
blood condition strongly suggested the existence of a myelogenous 
leukaemia. The increase of the red cells in this case amounted to 
almost double their original number. 

To estimate the extent of a blood crisis, the following examina- 
tions are necessary : 

a. A determination of the absolute number of red corpuscles. 

b. A determination of the ratio between the white and red cells. 

c. A determination of the ratio between the nucleated red and 
white cells, in dried specimens, by the aid of a quadratic ocular 
diaphragm. 

Example. — Supposing that in a given case 3,500,000 red corpus- 
cles are found in the cbmm., while the ratio of the white to the red 
corpuscles is 1 : 100, and that of the nucleated red to the white 
1:10; 3500 nucleated red corpuscles must hence be present in 
each cbmm. of blood — i. e., 1 for each 1000 of normal red cor- 
puscles. 

From the standpoint of prognosis it is noteworthy that a fatal 
issue may be expected whenever the number of red cells falls 
below 1,500,000 and normoblasts are absent. 

The Megaloblasts. — These are usually from two to three times as large 
as the normoblasts, but may attain even more extensive proportions 
(Ehrlich's gigantoblasts). They are provided with a relatively large 
centrally located nucleus, which is wide-meshed and which with the 
triacid stain is not colored nearly so deeply as the normoblastic 
nucleus. In some specimens, indeed, the affinity for methyl-green 
is so little marked that at first sight a nucleus can hardly be distin- 
guished. With those staining mixtures, on the other hand, which 
contain methylene-blue as base, it can always be fairly well made 
out. But owing to the fact that these cells are almost invariably 
polychromatophilic, the nucleus may at first be overlooked, as the 
polychromatic protoplasm appears in the meshes of the nucleus and 



70 THE BLOOD. 

sometimes differs but little in eolor from the chromatin. The inex- 
perienced not infrequently mistake such cells for large mononuclear 
leucocytes that are somewhat off-color ; the character of the nucleus, 
however, viz., its wide meshwork, should prevent this mistake. 

Mitoses in megaloblasts are also at times seen. 

Under normal conditions a few megaloblasts may be found in the 
blood of very young infants, and it is noteworthy that in the severe 
types of secondary anaemia megaloblasts are far more apt to occur 
in children than in adults. But even then they are rare. Accord- 
ing to Ekrlich, the presence of megaloblasts is evidence of a rever- 
sion of blood formation to the embryonic type and of grave prognostic 
import. He regarded their presence as indicative of essential 
pernicious anaemia ; and, as a matter of fact, they are here quite 
constantly met with and represent one of the most important features 
of the disease. They are rarely numerous, however, and there are 
cases in which they do not occur in the circulating blood. A few 
cases of pernicious anaemia have indeed been reported in which 
neither megaloblasts nor normoblasts could be demonstrated at any 
time (Ehrlich, Lazarus, Engel, Pane l ). 

The modern tendency is to regard the appearance of megaloblasts 
in the blood as evidence of an anaemia of unusual severity, and not 
as an indication of any one disease or of malignant degeneration. 
While they are undoubtedly most constant in pernicious anaemia, 
they may also be met with in other forms. They have been found 
in leukaemia, in lead-poisoning, and even in chlorosis. I have seen 
a few megaloblasts in v. Jaksch's pseudoleukemia infantum, and in 
a case of severe infantile anaemia referable to amoebic colitis. In 
cancer of the stomach, according to Osier and McCrae, they are 
rarely if ever found. Askanazy 2 has reported an interesting case 
of bothriocephalus infection in which the megaloblastic type of blood 
regeneration disappeared after expulsion of the parasites — sixty- 
seven in number — and was replaced by the normoblastic type, the 
case ending in recovery. 

In cases of traumatic anaemia unusually small nucleated red cells 
have at times been observed. These are termed mieroblasts. They 
have attracted but little attention and are quite rare. I have seen 
such cells, measuring not more that 3-3.5 [i, in a case of pernicious 
anaemia at the time of the blood crisis, in which large numbers of 
normoblasts were also present. 

1 Pane, " Sull' anemia progressiva mortale senza corpuscoli rossi, nueleati neJ 
sangue," Riform. med., 1900, No. 263. 

2 Askanazy, Zeit. f. klin. Med., 1895, vol. xxvii. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 



71 



The Leucocytes. 

General Characteristics. — The leucocytes, or white corpuscles 
of the blood, as seen in the wet preparation (Plate II., Fig. 1), are 
roundish or irregularly shaped cells, which vary in size, but for the 
most part are larger than the red corpuscles. They are all nucleated, 
and, as the term indicates, devoid of coloring-matter. In a general 
way they may be divided into two distinct classes, viz., those which 
are granular and those which are not granular. 

The granular cells are by far the most numerous, and are charac- 
terized by the fact that they are capable of active locomotion. Even 
without a warm stage it is almost always possible to observe this 
in the ordinary wet preparation. The moving cells at once attract 



Fig. 14. 




Phagocytosis. 

attention by their irregular outline. On careful examination with a 
high power it will then be noted that the cell advances in a definite 
manner, which is quite analogous to what is seen in the amoeba. 
The protoplasmic portion of the cell manifestly consists of two 
parts, viz., a non-granular hyaline ectosarc and a granular endosarc. 
As the leucocyte progresses the hyaline ectosarc advances with a 
flowing motion, forming a distinct layer in front of the granular endo- 



72 THE BLOOD. 

sarc, which itself then merges into the non-granular portion. The 
moving leucocyte is roughly pear-shaped, with the base in advance, 
while the rear end tapers markedly and frequently seems to drag 
behind it a small roundish mass which, like the main body of the 
cell, is also granular. These granular leucocytes are the so-called 
phagocytes of Metchnikoff, so termed from the fact that, like amoebae, 
they take up foreign matter into their interior. This can be de- 
monstrated especially well in malarial blood taken at the time of 
a paroxysm, when extracellular organisms (page 188) are present. 
Placing one of these in the centre of the field, it may be observed 
how one or more leucocytes will gradually approach the organism 
and finally engulf it, as shown in the accompanying illustration 
(Fig. 14). According to Metchnikoff, the phagocytic function is 
the most important function of the leucocytes, and the outcome of a 
bacterial invasion, figuratively speaking, will depend upon the 
superiority of the organisms engaged in warfare. 

The nucleus of the granular leucocytes is polymorphous — i. <?., it 
is composed of different lobes which in life are joined together, w r hile 
in dead cells the impression is gained as though a number of nuclei 
existed ; this fact accounts for the earlier but more common term 
polynuclear, instead of polymorphonuclear leucocytes. 

While the granules in the majority of the leucocytes are very fine 
(Plate II., Fig. 1), on careful search some cells will be found in 
which they are very coarse and highly refractive. This coarsely 
granular variety is very characteristic in appearance and at once 
attracts attention. The cells are far less numerous, however, and 
as a matter of fact represent only from 1 to 4 per cent, of the total 
number of the leucocytes, while the finely granular variety represents 
from 60 to 70 per cent. 

The non-granular leucocytes, in contradistinction to the granular 
variety, are all mononuclear. They are quite hyaline in appearance, 
and are readily overlooked by the beginner unless a somewhat sub- 
dued light is used in the examination. Two varieties may be recog- 
nized : one about the size of a red corpuscle, the other somewhat 
larger. The nucleus in both varieties occupies a considerable por- 
tion of the cell and is surrounded by a layer of protoplasm which is 
practically hyaline. Every cell, it is true, contains a few granules 
collected at a certain point along the periphery, where the proto- 
plasm is more extensively developed than elsewhere ; but these 
granules, in contradistinction to those which we see in the poly- 
nuclear varieties, probably represent nodal points in the cytoreticulum, 
and not a specific secretory product, as which Ehrlich and his school 
view the granules of the polynuclear variety. In the small mono- 
nuclear form one or sometimes two small brownish granules can also 
usually be discerned somewhere in the peripheral layer of the proto- 
plasm. Of the significance of this granule, so far as I am aware, 



PLATE IV. 





n 



"**&•. 



L.S. 



Lymphocytes. 

The cell a shows nucleus after division, each with a nucl 



eolus. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 73 

nothing is known, nor has its presence been previously described 
(see Plate II., Fig. 1). 

The non-granular mononuclear leucocytes, in contradistinction to 
the polynuclear granular variety, were formerly regarded as non- 
motile. Jolly, Wolff, and others have recently shown, however, 
that they also are capable of changing their form even though pro- 
gressive locomotion may not occur. The change in form can readily 
be demonstrated even without a warm stage, and it will be observed 
that the nucleus takes an active part in these changes. Heinen 
demonstrated this in my laboratory without having knowledge of 
Wolff's paper, which then had just appeared. 

Classification. — While it is possible to distinguish several varie- 
ties of leucocytes in the wet and unstained preparation, a more com- 
plete picture of the structure of the individual forms may be obtained 
from a study of specimens that have been variously stained with 
acid, basic, and neutral anilin dyes (see page 119). The study of 
such preparations, moreover, forms the most satisfactory basis of 
classification of the different forms of leucocytes which we possess 
at the present time. We distinguish the following varieties : 

1. The Lymphocytes (Small Mononuclear Leucocytes) (Plate IV.). — 
The lymphocytes which occur normally in the blood are for the most 
part a little smaller than the red corpuscles or of equal size. The 
nucleus is single and surrounded by a narrow rim of protoplasm which 
is generally described as non-granular. But, as I have pointed out, a 
few granules can almost always be made out in the wet preparation 
at a certain point along the periphery where the protoplasm is a 
little more extensively developed. These granules, however, prob- 
ably represent nodal points of the cytoreticulum, and are not to be 
regarded as in any way analogous to the granules which are met 
with in the polynuclear leucocytes. Nucleus and protoplasm are 
both basophilic, and, generally speaking, the protoplasm is so more 
markedly than the nucleus. This is best seen in specimens that 
have been stained with methylene-blue, where the lymphocytes for 
the most part present a comparatively feebly staining nucleus which 
is surrounded by a rim of dark blue. Other cells belonging to the 
same group, however, will also be seen in which this is not marked, 
but in which the staining affinities of both nucleus and protoplasm 
appear about the same. These cells are generally a little larger 
than the first variety, with a somewhat broader zone of protoplasm 
and an excentric position of the nucleus. We may speak of them 
as medium-sized lymphocytes in contradistinction to the small-sized 
variety. A still larger form may also be met with, but it is not 
commonly seen under normal conditions except in children. The 
staining properties of these large lymphocytes are essentially the 
same as those of the smaller varieties ; in some the protoplasm is 
more intensely basophilic than the nucleus, while in others the 



74 THE BLOOD. 

basophilia of both is about the same. The position of the nucleus 
may be either concentric or excentric, as in the smaller forms. 
This large type is notably seen in acute lymphatic leukaemia, but it 
also occurs in the chronic form. 

With certain dyes, like methylene-blue, the protoplasm of the 
lymphocytes does not appear perfectly homogeneous, but presents a 
peculiar granular appearance. This is referable to nodal points of 
the cytoreticulum and does not represent a true granulation. With 
methyl-green, and hence with Ehrlich's triacid stain, the protoplasm 
is perfectly homogeneous and appears as a pale rim about the some- 
what more deeply staining nucleus. While it is thus impossible 
with the usual dyes to demonstrate the existence of a true granula- 
tion in the lymphocytes, Michaelis 1 has called attention to the fact 
that with eosin-methylene-azure solutions (page 123) distinct granules 
can be seen. Their significance, however, has not as yet been 
established. Very curiously these granules could not be demon- 
strated in the lymphocytes obtained from the lymph-glands directly, 
and it appears that they are present in only a certain percentage of 
those occurring in the blood. The number of granules in a cell is 
variable ; in some only two or three are seen, while in others the 
protoplasm is literally studded with them. Their size varies between 
that of the common neutrophilic and that of the eosinophilic varieties. 

In wet specimens, as I have pointed out, one or two reddish- 
brown granules are quite commonly seen in most of the lymphocytes. 
In stained preparations these cannot be demonstrated. 

The outline of the cell in the smaller forms is usually fairly 
smooth, but in the larger varieties it is often shaggy, and at times 
specimens are seen with a number of distinct knobs. These mani- 
festly represent bits of protoplasm which are being constricted off, a 
process which can be directly observed in the wet preparation. In 
the dried and stained specimen also it is not uncommon to meet with 
such particles occurring free in the blood, the origin of which is 
quite manifest from their staining properties, which are the same as 
those of the parent cell. 

The nucleus, in the smaller forms especially, is concentrically 
located, while in the larger varieties, in which the protoplasm is 
more extensively developed, it commonly occupies an excentric 
position. In the stained specimens, especially in the larger cells, it 
is sometimes surrounded by a faint areola, which is probably owing 
to artificial retraction. The nucleus is more commonly oval or bean- 
shaped than round ; deep invaginations are not often seen and frag- 
mentation of the nucleus is rare. Such cells present an appearance 
which is altogether different from that of the true polynuclear 
elements. 

Lymphocytes undergoing mitosis are sometimes seen in the blood 

1 Michaelis and Wolff, Virchow's Archiv, 1902, vol. clxvii. p. 151. 



PLATE V. 



/~N 



<0 





1 




Note the size of the various leucocytes, as compared with the red corpuscles at 15. Figs. 
1, 2 and 6 represent the most common forms of the small type of lymphocytes ; 3 and 5 belong 
to the same group, but are manifestly atypical ; 3 shows the knob-like projections, described 
in the text ; 4 represents the large type of the lymphocyte, and shows the vacuolated appear- 
ance of the protoplasm, which is so commonly seen. The metachromatism of the protoplasm, 
however, does not appear here as in nature. 7 and 8 are representatives of the large variety 
of mononuclear leucocytes ; 9 may be classed as a transition form, which is as yet devoid of gran- 
ules ; 13 represents a neutrophilic myelocyte, 14 an eosinophilic myelocyte, 10 a neutrophilic 
polynuclear leucocyte, 11 an eosinophile of the same type, and 12 a typical basophilic leucocyte. 

The preparations were stained with the eosinate of methylene-blue and drawn to scale. 
fBausch and Lomb, Eye-piece 1 inch, objective i-i2th.) 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 75 

of lymphatic leukaemia. Characteristic figures, however, are com- 
paratively rare, and it is more common to meet with cells in which 
division of the nucleus has already occurred (Plate IV.). Nucleoli 
are not usually seen in the lymphocytes of the normal blood, and 
seem to be comparatively infrequent also in the blood of lymphatic 
leukaemia. Occasionally, however, specimens are met with in which 
they are quite distinct, and at the same time multiple ; in such cases 
active cell-division seems to take place in the circulating blood. 

The reaction of the protoplasm of the lymphocytes, as tested with 
the erythrosin method (page 139), is strongly alkaline. The cells 
contain no glycogen. 

In adults the number of the lymphocytes normally varies between 
20 and 30 per cent. Higher values are found in young children, 
especially during the first year of life, when the lymphocytes con- 
stitute from 50 to 60 per cent, of the total number. At birth, how- 
ever, they are curiously less numerous than in adult life, viz., only 
about 15 to 16 per cent. They very soon increase, however, and 
by the twelfth day it is usual to have from 40 to 50 per cent. 
After the fifth year adult values are normally the rule. 

In disease the number of the lymphocytes may be increased or 
diminished, conditions which are spoken of respectively as lympho- 
cytosis and lymphopenia (see pages 105 and 107). 

While it was formerly supposed that the lymphocytes originate 
only in the lymph-glands proper, there is evidence that they may 
be formed wherever there is lymphoid tissue, and hence also in the 
spleen and in the bone-marrow. They are probably derived from 
the so-called large lymphocytes of the germinal centres indirectly 
through a process of differentiating karyokinesis, and represent fully 
differentiated cells which are incapable of further development. 

The large lymphocyte itself, as I have pointed out, practically 
occurs in the blood only under pathological conditions. According 
to Pappenheim's views, it represents the Ur or Stammzette-, from 
which all other forms of leucocytes are directly or indirectly derived. 
The large lymphocytes are identical with Benda\s lymphogonia, 
Troje's lymphoid marrow-cells, NagelFs myeloblasts, and the undif- 
ferentiated lymphoid cell of Michaelis and Wolff. 

2. The Large Mononuclear Leucocytes (Plate V.). — These are mostly 
two or three times as large as the red corpuscles and provided with a 
large single nucleus, which is surrounded by a relatively wide zone 
of non-granular protoplasm. The nucleus in some cells is oval or 
elliptical, while in others it is more or less invaginated. In the 
past it has been customary to classify large mononuclear leucocytes 
with karyolobic nuclei under a special heading — the transition-forms 
of Ehrlich — and it was supposed that a certain number of large 
mononuclear leucocytes aged directly in the circulating blood into 
the polynuclear neutrophilic leucocytes with the transition-form as 



76 . THE BLOOD. 

an intermediary stage. This view, however, is open to many 
objections and there are good grounds for the present tendency to 
regard the transition-form merely as the mature form of the large 
mononuclear leucocyte and as incapable of further differentiation 
(Pappenheim). 

In the wet preparation the large mononuclear leucocytes are 
exceedingly hyaline, so that they are readily overlooked by the 
beginner. Both nucleus and protoplasm are basophilic, but much 
less markedly so than the lymphocytes, and it is noteworthy that 
the protoplasm usually possesses a less marked affinity for the basic 
dye than the nucleus. Cells, however, are also met with in which 
the affinity for the dye is about the same in both structures. If by 
chance this occurs in specimens which are somewhat smaller than 
usual, a certain amount of difficulty arises in differentiating such 
small " large " mononuclear leucocytes from certain lymphocytes, in 
which the staining reaction is also not typical. A hard-and-fast 
line of distinction cannot here be drawn, and in every differential 
leucocyte count the personal equation will of necessity enter into 
consideration. The salient characteristics of the two types should, 
however, be borne in mind ; in the lymphocytes the protoplasm is 
but feebly developed in relation to the size of the nucleus, while in 
the large mononuclear leucocyte the reverse holds good. The proto- 
plasm in the latter, moreover, is apparently much more delicate in 
structure, and is readily wrinkled by contact with adjacent cells ; 
not infrequently cells of this type are found which have manifestly 
been torn or otherwise injured during the preparation of the speci- 
men, while the lymphocytes are usually well preserved. 

In preparations that have been stained with Ehrlich's triacid both 
nucleus and protoplasm are very faintly colored and the latter 
appears perfectly homogeneous ; but in specimens which have been 
stained with mixtures containing methylene-blue as the basic com- 
ponent, the protoplasm presents a somewhat granular appearance, 
which, as in the lymphocytes, is referable to the existence of a cyto- 
reticulum. A certain proportion of the large mononuclear leucocytes 
(including the transition-forms), however, also contain granules 
which, as in the case of the lymphocyte, can be stained with eosin- 
methylene-azure solutions. 

Inclusive of the transition-forms the large mononuclear leucocytes 
normally represent from 1 to 6 per cent, of the total number. They 
are relatively more numerous in young children, in whom the highest 
values are found between the sixth and ninth day after birth. 
Many of the cells at this time are of the type of the transition-form ; 
they may number 18 per cent. 

According to Ehrlich, the large mononuclear leucocytes originate 
in the bone-marrow and possibly also in the spleen. As Pappenheim 
has suggested, they may develop directly, cytogenetically, from the 



PLATE VI 








*IV 








L.S. 



Granulocytes. 



a, polynuclear neutrophilic leucocytes ; 5, polynuclear eosinophilic leucocytes ; 
c, mast cells ; d y young eosinophilic myelocytes ; e, neutrophilic myelocytes ; /, the 
nucleus here has just undergone division : the clear space is a vacuole. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 77 

" large " lymphocytes, and then age into the transition-form, which 
represents the final stage in the development of this type. The 
former view, according to which the large mononuclear leucocyte 
develops directly cytogenetically from the small lymphocyte, has 
practically been abandoned. 

3. The Neutrophilic Polynuclear Leucocytes (Plate VI.). — These 
cells are a little smaller than the large mononuclear leucocytes and 
the transition-forms and represent the finely granular variety, already 
referred to. They are the phagocytes, xar'i£;o%yv y and as such cap- 
able of most active progressive locomotion. The nucleus is a long 
body, which is commonly twisted upon itself in various ways, so as 
to resemble the letters S, Y, E, and Z. Often also it is broken up 
into fragments, suggesting the presence of several nuclei, which 
accounts for the original designation of these forms as polynuclear 
leucocytes. As Ehrlich has suggested, however, the polynuclear 
appearance is probably referable to post-mortem changes, the condi- 
tion of the nucleus being in reality polymorphous. They are accord- 
ingly also spoken of as polymorphonuclear neutrophilic leucocytes ; 
but the older term, though not so correct, is the more common. 
The nucleus stains very readily with all nuclear dyes ; it is coarsely 
reticulated and generally shows evidence of a central nodal thickening 
in each one of its lobes. 

The protoplasm proper has a marked affinity for most of the 
acid dyes ; its reaction with the erythrosin method is alkaline, but 
less so than in the lymphocytes and large mononuclear leucocytes. 
Under normal conditions a glycogen reaction is not obtained. 

Embedded in the protoplasm are numerous fine granules — the 
e-granulation of Ehrlich — which are characterized by their affinity 
for neutral dyes. Hence the term polynuclear neutrophilic leucocytes. 
These granules are ordinarily very abundant ; but in disease they 
may diminish in number until very few are left, and in certain 
cases of chronic leukaemia they may indeed be absent altogether. 
Ewing 1 has called especial attention to the decrease in the number 
of the granules in the acute leucocytoses ; this decrease, in my 
experience, is even more marked in the chronic cases, and especially 
so in the septic infections. Associated with the diminution in the 
number of the granules there are very frequently also certain degen- 
erative changes affecting the nuclei. These may be of the type of 
karyolysis with swelling and loss of chromatin, or of karyorhexis 
with hyperchromatosis and fragmentation of the nucleus. The 
former is the more usual in the acute leucocytoses, while the latter is 
seen especially in leukaemia. In cases of the myelogenous variety 
it is here quite common to note complete fragmentation of the 
nucleus into six to ten segments. This phenomenon was first 
observed by Ehrlich in a case of hemorrhagic smallpox, and is of 
1 Ewing, Clinical Pathology of the Blood, Lea Bros., 1st ed., p. 113, 



78 THE BLOOD. 

common occurrence in fresh exudates. Cell degeneration associated 
with loss of chromatin and swelling, while it no doubt occurs to a 
greater degree in disease, may also be observed under normal condi- 
tions. In every dried and stained specimen a certain number of 
such cells will be found in which the nucleus appears as a much 
swollen and but faintly staining shadow, the Kemschatten of the 
Germans, sometimes surrounded by some of the granules, which 
appear scattered as though the cell had been burst asunder by force ; 
at other times the Kemschatten alone remains and nothing is seen 
of the body of the cell. 

I have stated that the loss of granules on the part of these cells 
may go on to a point where they are absent altogether. It may 
happen, however, that the granules are only apparently absent, and 
merely do not react as usual with ordinary dyes. A proper explana- 
tion of this peculiar behavior cannot be given, but every worker 
in blood is no doubt familiar with the phenomenon. Sometimes a 
change in the mode of fixation will cause the granulation to appear ; 
at other times it may be demonstrated by the aid of some other dye. 

Vacuolization of the polynuclear leucocytes is. very much less 
common than in the case of the mononuclear elements. 

Neusser l some years ago called attention to the fact that with a 
certain modification of Ehrlich's triacid stain it is possible to demon- 
strate the presence of basophilic granules about the nucleus of some 
of the polynuclear leucocytes, as well as the mononuclear elements. 
He, as well as Kolisch, 2 regarded the presence of these perinuclear 
granules as characteristic of the so-called uric acid diathesis. As 
tubercular disease, moreover, is usually not seen in such cases, Neus- 
ser thought the presence of these granules in cases of phthisis to be 
a favorable symptom. Futcher, 3 on the other hand, was unable to 
confirm these observations, and my own investigations 4 are likewise 
opposed to Neusser's conclusions. I was able to demonstrate the gran- 
ules both in health and disease in almost every case, and was at one 
time even led to think that their absence was of more significance than 
their presence. A relation between their presence and the elimina- 
tion of uric acid or xanthin bases certainly does not exist. Within 
recent years the subject has received no further attention, especially 
since Ehrlich expressed the belief that the granules are artefacts. 
He states that they are only exceptionally seen when solutions of 
chemically pure crystalline dyes are used, from the Actiengesellschaft 
fur Anilinfarbstoffe in Berlin. 

The neutrophilic granulation, in contradistinction to the eosino- 
philic granulation and the mast-cell granulation, does not occur 
generally distributed among vertebrate animals, but, according to 

1 Neusser, Wien. klin. Woch., 1894, p. 71. 

2 Kolisch, Ibid., 1895, p. 797. 

3 Futcher, Johns Hopkins Hosp. Bull.. May, 1897. 
* Simon, Am. Jour. Med. Sci., vol. cxvii. p. 139. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 79 

Ehrlich, is found only in man and the ape. My own researches in 
this direction tend to confirm Ehrlich's views, which have been repeat- 
edly assailed from several directions. Granulations of this character 
which are not common to all animals are termed special granulations. 

The poly nuclear neutrophilic leucocytes are derived from corre- 
sponding mononuclear forms — the neutrophilic myelocytes — which 
are normally found only in the bone-marrow. They result from 
these directly and represent their adult form. Any other origin for 
the polynuclear neutrophiles has not been satisfactorily established. 
Ehrlich, it is true, has admitted that a certain number of the cells 
may develop in the circulation from the large mononuclear leuco- 
cytes with the so-called transition -forms as intermediate stages ; but, 
as I have pointed out, there is a strong tendency at present to 
regard the transition-form as the terminal stage in the development 
of the large mononuclear leucocyte and to separate this group 
altogether from the granular varieties. There is an ontogenetic rela- 
tionship between the two, but not a cytogenetic relation. 

The polynuclear neutrophiles are the most common leucocytes of 
the blood and normally constitute from 60 to 70 per cent, of the 
total number. In young children they are relatively less numerous 
excepting during the first twenty-four hours of life, when they may 
number 73 per cent. But they rapidly diminish, so that values of 
from 20 to 40 per cent, may be regarded as normal during the first 
year. Low values continue practically to the twelfth year, though 
the numbers gradually rise. From the twelfth to the fourteenth year 
60 per cent, may be regarded as an average ; after that age the values 
given for the adult hold good. 

A pathological increase in the number of the polynuclear neutro- 
philes constitutes the most common form of hyperleucocytosis (see 
page 86). 

4. The Polynuclear Oxyphilic or Eosinophilic Leucocytes (Plate VI.). 
— In size and general appearance these cells resemble the polynuclear 
neutrophiles, and like these are capable of progressive locomotion and 
phagocytosis. The granules — the a-granulation of Ehrlich — how- 
ever, are much larger and highly refractive, and possess a marked 
affinity for acid dyes, such as acid fuchsin and eosin. Hence the 
term oxyphilic or eosinophilic leucocytes. With neutral dyes or basic 
dyes they will not stain. The appearance of the individual granules 
varies somewhat in stained preparations. Some are round, others 
oval ; some appear to stain throughout, others make the impression 
of little vesicles with a limiting membrane, which alone takes the 
dye, while the interior remains unstained. By means of MacCal- 
lum ? s method Barker 1 has shown that the granules contain iron. 
They are insoluble in ether and cannot be stained with osmic acid. 
They are therefore not composed of fat. 

1 Barker, Johns Hopkins Hosp. Bull., 1894, p. 93. 



80 THE BLOOD. 

The protoplasm of the eosinophilic leucocytes is slightly baso- 
philic. The nucleus is usually bilobed, sometimes trilobed, and in 
stained specimens it is quite common to find the individual lobes 
unconnected by threads of chromatin ; often the two lobes are 
situated at opposite poles. As a rule the nucleus is less markedly 
basophilic than that of the neutrophilic variety. 

The same degenerative changes which have been described in con- 
nection with the poly nuclear neutrophiles may also be observed in 
the eosinophils, and here, as there, one can at times note a material 
diminution in the number of the granules. I have never observed 
their entire absence, however, and it is noteworthy that in those 
cases of chronic leukaemia in which the neutrophilic granulation may 
disappear the eosinophilic variety remains. 

Under normal conditions the percentage of the eosinophils varies 
between 2 and 4, corresponding, according to Zappert, to minimum 
absolute values of 50—100 and maximum absolute values of 200- 
250, with 100-200 as an average. 

While repeated attempts have been made to connect the eosino- 
philic leucocytes of the blood cytogenetically with the neutrophilic 
variety, there is insufficient evidence to support this view. On 
the other hand, there are strong reasons for believing with Ehrlich 
that, analogous to the neutrophilic variety, the polynuclear eosino- 
phils are normally formed in the bone-marrow, and here only from 
mononuclear eosinophilic cells — the eosinophilic myelocytes. 

Unlike the neutrophilic granulation, the oxyphilic variety is repre- 
sented in all vertebrate animals, and in birds may occur in the form 
of crystalloids. 

5. The Mast-cells (Polynuclear Basophilic Leucocytes) (Plate VI.). 
— The mast-cells which are normally found in the blood are approxi- 
mately of the same size as the polynuclear neutrophiles and eosino- 
philes. In myelogenous leukaemia, however, in which they are espe- 
cially numerous, the size is more variable ; on the one hand, they may 
only measure 3.5 /i in diameter, while on the other they may attain 
a dimension of 22 /i. The nucleus is polymorphous ; but the ten- 
dency to form individual lobes is far less marked than in the corre- 
sponding eosinophilic and neutrophilic elements. Its affinity for basic 
dyes is quite feeble, so that it is often difficult in stained preparations 
to make out the boundary-line between nucleus and protoplasm. It 
is almost always excentrically located and usually has a fairly uniform 
diameter of 4 p.. In the smaller specimens the nucleus occupies 
almost the entire cell and is covered by a very thin layer of granules. 

Embedded in the protoplasm lie granules of variable size — the 
^-granulation of Ehrlich — some of which are fully as large as the 
eosinophilic granules, while others are much finer. They are 
characterized by their affinity for basic dyes and the fact that with 
certain ones they stain metachromatically, viz., in a color which is 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 81 

different from that of the dye itself, which latter must be simple and 
not compound. Tissue elements which will stain in this manner are 
spoken of as chromotropic elements. Only a limited number of 
dyes have metachromatic properties. The most notable ones are the 
violet basic dyes hexamethyl-violet, cresyl- violet, thionin, neutral 
violet, and amethyst-violet ; further, the blue dyes methylene-azure 
and toluidin-blue, and the red basic dyes pyronin, amidin-red, 
neutral red, and saffranin. With the latter group the mast-cell 
granules are colored yellow ; with most of the violet dyes red, and 
with cresyl-violet R (extra) almost a pure brown. Methyl-green 
does not stain the mast-cell granules unless it is contaminated with 
methyl-violet, and for this reason the granules remain colorless in 
specimens that have been stained with Ehrliclr's triacid stain. 

The mast-cell granules are absolutely basophilic, viz., they can 
only be stained with basic dyes, and retain the basic dye on subse- 
quent differentiation in acid media. They are capable, moreover, 
of taking up the basic dye from its acidified solutions, as in the case 
of Ehrlich's dahlia-acetic acid mixture (see page 136). 

The granules of the common mast-cells of normal blood are quite 
resistant to water, while in myelogenous leukaemia cells are met with, 
the granules of which dissolve with great readiness (Michaelis). Their 
chemical nature is still a matter of dispute, but there is a tendency to 
associate the mast-cell with the formation of mucin. This, however, 
presupposes the identity of the blood mast-cell with the common 
mast-cell of connective tissue. In the past this has been tacitly 
assumed, but Pappenheim more especially has called attention to the 
fact that the hematogenic mast-cell differs from the histogenic form, 
and that the two probably represent different species. 

The mast-cell apparently occurs in the blood of all vertebrate 
animals. In human blood their number rarely exceeds 0.5 per cent. 
Ewing states that he constantly failed to find mast-cells in the better 
class of healthy subjects, while in hospital and dispensary cases with 
minor ailments they appeared to be more numerous. My own 
observations do not bear this out ; in my experience they are 
invariably present in health irrespective of the general nutrition 
of the individual. 

The origin of the mast-cells of the blood has not been definitely 
ascertained. Ehrlich supposed that they originated from the con- 
nective-tissue cells as the result of hypernutrition, while Harris 
suggests that they may be derived from the large mononuclear 
leucocytes. According to Pappenheim, the mast-cell originates in 
the bone-marrow from a granular mononuclear type which corre- 
sponds to the eosinophilic and neutrophilic myelocytes. 

6. The Myelocytes. — The myelocytes are mononuclear granular 
cells, which are normally not found in the circulation, but are 
encountered only in the bone-marrow. 

6 



82 THE BLOOD. 

Generally speaking, they represent the juvenile form of the poly- 
nuclear leucocytes of the blood, and we accordingly distinguish three 
varieties, viz., the neutrophilic, the eosinophilic, and the basophilic 
myelocytes. The last two named varieties, according to our present 
ideas, age directly into the corresponding polynuclear forms — L e., they 
become the common eosinophiles and the mast-cell. In the case of 
the neutrophilic variety it appears that two types exist, a smaller 
and a larger form, which Pappenheim 1 designates respectively as the 
trachychromatic and the am bly chromatic form. These are onto- 
genetically derived, the first from the last, but only the trachychro- 
matic variety ages into the common polynuclear neutrophiles of the 
circulating blood. The nucleus of the amblychromatic form as it 
matures likewise becomes polymorphous, but normally the cell 
remains an inhabitant of the bone-marrow even then. 

As regards the origin of the myelocytes in turn, I incline toward 
Pappenheim's idea, according to which all three varieties result from 
the large lymphocytes through a process of heteroplastic differentia- 
tion. 

a. The Neutrophilic Myelocytes. — These, as I have stated, 
are of two kinds. The one type, the amblychromatic myelocyte of 
Pappenheim, is a large cell provided with a relatively large cen- 
trally located round nucleus which stains but feebly with basic dyes. 
This is surrounded by a comparatively narrow zone of protoplasm 
which contains very fine neutrophilic granules. As the cell matures 
the nucleus becomes smaller and indented, so that forms result such 
as those pictured in Plate VI. ; the position of the nucleus is 
then usually excentric. The protoplasm at the same time becomes 
relatively more abundant. 

The second type, viz., the trachychromatic myelocyte, is a smaller 
cell, which is essentially characterized by the fact that its nucleus 
stains quite intensely with basic dyes. The protoplasm is faintly 
oxyphilic and the granulation rather coarser than in the amblychro- 
matic variety. As this cell matures the protoplasm becomes rela- 
tively more abundant and the nucleus distinctly polymorphous ; it 
then constitutes the common polynuclear neutrophile of the circu- 
lating blood. 

Neutrophilic myelocytes undergoing mitosis are sometimes seen 
in the circulating blood in cases of myelogenous leukaemia ; on the 
whole, however, they are rare, and it is more common to meet with 
cells in which the division of the nucleus has already taken place 
(Plate VI.). 

Miiller and Jolly have shown that the neutrophilic myelocytes of 
the circulating blood are capable of active locomotion. 

b. The Eosinophilic Myelocytes. — In the more mature 
forms the color of the eosinophilic granulation -on staining with 

1 A. Pappenheim, Virchow's Archiv, vols. clix. and clx. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 83 

eosin-methylene-blue mixtures is a pure eosin-red. The younger 
forms, however, present a purplish-violet color, and some granules 
may indeed be seen which are a pure blue (Plate VI.). This appearance 
is owing to the fact that the young eosinophilic granule is physically 
cyanophilic and chemically amphophilic, whereas the mature granule 
is physically erythrophilic, but chemically absolutely oxyphilic. This 
is well shown by staining such young cells with a mixture in which 
the basic dye is of a light color and the acid component dark, such as 
vesuvin on the one hand and water-blue on the other. The mature 
eosinophilic granules will then take on the blue color of the water- 
blue, while the young granules which stained blue with the eosin- 
methylene-blue mixture, and which we might accordingly have 
regarded as basophilic, are now likewise colored by the acid blue 
instead of the basic vesuvin, thus showing that they are in reality 
not basophilic, but amphophilic-cyanophilic. 

The protoplasm of the eosinophilic myelocytes is basophilic. 

The size of the cells is, quite variable; some are considerably 
larger than the corresponding polynuclear form, while others are much 
smaller. The cyanophilic cells are, generally speaking, the largest. 

According to the observations of Midler and Jolly, the eosino- 
philic myelocytes are capable of progressive locomotion. 

c. The basophilic myelocytes, like the eosinophilic and neutro- 
philic varieties, may be of variable size and are provided with a 
large centrically located nucleus, which is often distinguished only 
with difficulty from the surrounding protoplasm. 

7. Ehrlich's Neutrophilic Pseudolymphocytes. — These bodies, accord- 
ing to Ehrlich, are produced by direct division of the polynuclear 
neutrophilic leucocytes. They are about as large as the small 
lymphocytes and provided with a single deeply staining nucleus, 
which is surrounded by a narrow zone of protoplasm containing 
neutrophilic granules. They differ from the small trachychromatic 
myelocytes essentially in their small size. They are rarely seen. 
Ehrlich states that he first observed them in a case of hemorrhagic 
smallpox, but that they also occur in recent pleural effusions. The 
cells no doubt are degeneration-forms and do not represent a sep- 
arate species. 

8. Irritation Forms. — These are mononuclear non-granular cells, 
the protoplasm of which is stained a rich brown by the triacid 
mixture. The nucleus is round, often excentrically located, and 
colored a bluish green. The smallest forms are somewhat larger 
than the lymphocytes. According to Turk, who first described these 
cells, they are met with under the same conditions as the myelocytes. 
Possibly they represent an early stage in the development of the 
nucleated red corpuscles. 

Iodophilia. — On staining blood-smears of normal individuals with 
iodine (see page 138) the protoplasm of the leucocytes is colored a 



84 THE BLOOD: 

bright yellow, while the nucleus is somewhat refractory and takes on 
a lighter tint. Under certain pathological conditions this staining 
quality is modified ; cells are then seen in which reddish-brown 
granules appear in the protoplasm or it may occur that this presents 
a diffuse brownish color throughout. This intracellular reaction 
affects the polynuclear neutrophils almost exclusively ; the mono- 
nuclear elements may, however, also react, in which case one com- 
monly sees large pale-brown granules arranged about the nucleus in 
a single row. In eosinophiles the reaction does not occur. The 
extent to which the leucocytes are involved is quite variable ; in 
some cases a few cells only are affected, while in others one is scarcely 
able to find a normal cell in an entire preparation. 

An extracellular reaction also occurs, but is of little clinical in- 
terest, as it is not infrequent even in health ; it occurs in small 
roundish or oval masses, which are possibly true plaques, but which 
may also be small Jbits of protoplasm derived from leucocytes. 

As to the nature of the substance which reacts with the iodine in 
the manner indicated, there is no uniformity of opinion. Ehrlich 
regards it as glycogen, and assumes that this is present normally in 
every cell in the form of a colorless compound, from which the 
free glycogen is under certain conditions split off, and can then be 
demonstrated as such. Czerny, on the other hand, looks upon the 
iodophilic substance as an antecedent of amyloid, while Goldberger 
and Weiss view it as peptone. Kaminer has shown that normal 
bone-marrow does not contain iodophilic leucocytes, but that they 
may here be found when they are present also in the blood. He 
concludes that the reaction is a degenerative phenomenon and not an 
evidence of regeneration. 

The occurrence of the reaction in disease has been studied especially 
by Gabritschewsky, Czerny, Livierato, Kaminer, Cabot, and Locke. 
From these investigations it appears that septic conditions of all 
kinds furnish a positive reaction. Locke's list of diseases of this 
order includes general septicaemia, abscesses (excepting in the earliest 
stages), appendicitis accompanied by abscess formation, general peri- 
tonitis, empyema, pneumonia, pyonephrosis, salpingitis with severe 
inflammation or abscess formation, tonsillitis, gonorrheal arthritis, 
and acute intestinal obstruction where the bowel has become gangre- 
nous. Locke concludes that no septic condition of any severity can 
exist without a positive reaction. In puerperal sepsis also it is 
constant (Kaminer). In pneumonia with frank resolution it dis- 
appears in from twenty-four to forty-eight hours following crisis. 
In typhoid fever a positive reaction is not commonly obtained before 
the end of the second week, and it may indeed remain absent 
throughout the course of the disease. In the differential diagnosis 
between a serous and a purulent pleuritic effusion the absence of the 
reaction points to the former condition. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 85 

In contradistinction to chlorosis, pseudoleukemia, and the common 
forms of secondary anaemia of moderate intensity, iodophilic leuco- 
cytes are found only in the severer forms of anaemia, such as perni- 
cious anaemia, leukaemia (notably in acute cases), and the severe forms 
of secondary anaemia. 

In animals the reaction can be produced artificially by infection 
with the streptococcus, the staphylococcus, the bacillus pyocyaneus, 
L6ffler ? s bacillus, the anthrax bacillus, that of Friedlander, the bacillus 
coli communis, or the typhoid bacillus ; as also by means of ricin, 
abrin, and the diphtheria toxin. Following the injection of oil of tur- 
pentine, croton oil, mustard oil, and silver nitrate, the reaction may 
occur even though bacterial infection has been avoided. In man it 
is also said to occur following narcosis. 

Literature. — Ehrlich, Zeit. f. klin. Med., 1882, vol. vi. Gabritschewsky, Arch, 
f. exp. Path. u. Pharmak., 1891, vol. xxviii. Czerny, Ibid., 1893, vol. xxxi. Gold- 
berger u. Weiss, Wien. klin. Woch., 1897. Hof bauer, Centralbl. f. inn. Med., 1899. 
Livierato, Deutsch. Arch. f. klin. Med., 1894, vol. liii. Kaminer, Berl. klin. Woch., 
1899, p. 119 ; and Deutsch. med. Woch., 1899, p. 206. Cabot and Locke, Jour. Med. 
Eesearch, 1902, vol. vii. Locke, Boston Med. and Surg. Jour., 1902, p. 289. 

Leucocytosis. 

While the number of the red corpuscles is normally fairly con- 
stant, that of the leucocytes is subject to not inconsiderable variation. 
It is influenced by the age and sex of the individual, the process of 
digestion, menstruation, pregnancy, the blood-vessel from which the 
specimen of blood is taken, etc. Generally speaking, the number 
of the leucocytes varies between 3000 and 10,000, the exact num- 
ber, cceteris paribus, depending upon the state of nutrition of the 
individual. In ill-nourished persons low values are the rule, while 
maximum numbers are generally associated with a state of excep- 
tional vigor and good nutrition ; 5000-7000 may be regarded as 
average values. 

An increase in the number of leucocytes is met with under the 
most diverse conditions, both in health and disease. When transi- 
tory, it is commonly designated as leucocytosis. But, as Goldscheider 
has rightly suggested, it would be better to restrict this term to 
indicate the number of the leucocytes in a general way, and to speak 
of an increase as hyperleucocytosis, and of a decrease as hypoleucocy- 
tosis. For the latter condition the term leucopenia has also been 
suggested. 

Ehrlich distinguishes between two forms of hyperleucocytosis, 
viz., an active and a passive form, the active form involving an 
increase of the polynuclear elements, while in the passive form the 
mononuclear cells are increased. This classification is based upon 
the assumption of absence of the power of locomotion on the part of 
the lymphocytes more especially, as contrasted with the polynuclear 



86 THE BLOOD. 

leucocytes. In the light of recent investigations this distinction 
can, however, no longer be upheld, since we know that the lympho- 
cytes are not only capable of changing their form, but, like the 
poly nuclear elements, are also doubtless subject to the laws of 
chemotaxis. 

As any variety of the leucocytes may be either increased or 
diminished, it is convenient for practical purposes to consider both 
possibilities in connection with each of the five normal types. We 
accordingly recognize : 

la. A polynuclear neutrophilic hyperleucocytosis. 

16. A polynuclear neutrophilic hypoleucocytosis. 

2a. A polynuclear eosinophilic hyperleucocytosis. 

26. A polynuclear eosinophilic hypoleucocytosis. 

3a. A mast-cell hyperleucocytosis. 

36. A mast-cell hypoleucocytosis. 

4a. A large mononuclear hyperleucocytosis. 

46. A large mononuclear hypoleucocytosis. 

5a. A lymphocytosis. 

56. A lymphopenia. 

The term myelcemia, or, as I should suggest, myelocytosis, may be 
used to designate the appearance of myelocytes in the circulating 
blood, and in conformity with the three recognized forms we may 
speak of a neutrophilic, an eosinophilic, and a basophilic or mast- 
cell myelocytosis. 

Until quite recently the general tendency in clinical laboratories 
has been to lay especial stress upon absolute numbers of leucocytes, 
and to neglect the relative values of the individual forms. This 
should not be, and I cannot insist too strongly upon the importance 
of the relative count, which in many respects is far greater than a 
knowledge of the total number. For this reason also I have chosen 
the consideration of the subject of hyperleucocytosis on the basis of 
the classification just outlined. 

Polynuclear Neutrophilic Hyperleucocytosis. — This is the 
most common form of hyperleucocytosis, and, as the term indicates, 
principally affects the polynuclear neutrophiles. Exceptionally it 
may be associated with a polynuclear eosinophilia, as well as with a 
lymphocytosis ; but as a general rule both eosinophiles and lympho- 
cytes are diminished. This diminution is often not only relative, 
but absolute as well. In very marked cases of hyperleucocytosis of 
this type it is not uncommon to meet with a few myelocytes, which 
are then also of the neutrophilic variety ; this is especially the case 
in children in whom the bone-marrow reacts more readily to stimu- 
lation. Eosinophilic myelocytes, on the other hand, are but rarely 
seen. 

Clinically we must distinguish between an increase of the poly- 
nuclear neutrophiles which may occur in health and the common 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 87 

hyperleucocytosis which is observed in disease. We may accord- 
ingly speak of a physiological and a pathological form. 

Physiological Hyperleucocytosis. — A physiological increase in the 
number of the leucocytes is notably observed at birth, during the 
process of digestion, in pregnancy, in association with severe mus- 
cular exercise, following the use of cold baths, etc. 

Leucocytosis of the Newborn. — According to the experience of 
most observers, the number of the leucocytes at birth varies between 
10,000 and 23,000, of which over 70 per cent, are polynuclear 
neutrophiles. The number then falls and at the same time the 
lymphocytes increase. The curves of the two varieties cross between 
the sixth and the ninth day, and by the twelfth day the lymphocytes 
are in excess. From the end of the first month to the fourteenth 
year there are then a gradual increase of the neutrophiles and a 
decrease of the mononuclear elements (Carstanjen). During the 
first year the total number of the leucocytes varies between 10,900 
and 12,900 (Gundobin). 

Digestive Leucocytosis. — The increase in the number of the leuco- 
cytes which is observed during the process of digestion affects both 
the polynuclear elements and the lymphocytes, though especially the 
latter. The eosinophiles are relatively at least diminished (Bieder). 
The total increase rarely exceeds 3500 in normal adults, wmile in 
y r oung children it may be much more marked. Schiff l thus cites an 
instance in which 19,500 leucocytes were counted one hour after 
birth, 27,625 after the first meal, and 36,000 after the fourth meal. 
It is especially pronounced after a preliminary period of fasting and 
following a meal rich in proteids. The maximum increase is usually 
observed between the third and fourth hour. 

In cases in which a hyperleucocytosis exists from other causes, as 
in pregnancy, in inflammatory diseases, etc., digestive hyperleuco- 
cytosis does not occur. In a few isolated instances it has also been 
found absent in apparently normal individuals without assignable 
cause. Under pathological conditions its absence is not uncommon, 
even though hyperleucocytosis referable to other factors may not 
exist. This is notably the case in carcinoma of the stomach, and it 
was once thought that, the absence of digestive hyperleucocytosis in 
doubtful cases could be interpreted as evidence in favor of its exist- 
ence. 2 Generally speaking, this is true even now, and we may say 
that in about 90 per cent, of all cases of carcinoma of the stomach 
digestive hyperleucocytosis does not occur. The symptom, however, 
is not pathognomonic, as a number of instances of carcinoma have 
been reported in which there was a distinct increase, and as digestive 
leucocytosis may also be absent in other conditions. In anaemic 

1 Schiff, Zeit. f. Heilk., vol. xi. p. 30, and 1890, p. 1. 

2 Schneyer, "Das Verhalten d. Verdauungsleukocytose b. ulcus rotundum u. carci- 
noma ventriculi," Zeit. f. klin. Med., vol. xxvii. p. 249. 



88 THE BLOOD. 

individuals, from whatever cause, especially large amounts of pro- 
teids are sometimes necessary to elicit an increase of the leucocytes 
(Muller 1 ) and in some cases a subnormal number may even be 
encountered (Eieder 2 ). 

To study digestive hyperleucocytosis, it is well to proceed as 
follows : 

a. The first blood-count should be made after the patient has 
fasted for about seventeen hours. 

b. After this period he receives a test-meal consisting of from 
200 to 1000 c.c. of milk, and one or two eggs, the amount varying 
with the condition of the patient. 

c. Further blood-counts are made one, two, three, and four hours 
later. 

d. The existence of a digestive hyperleucocytosis should only be 
regarded as proved if an increase of at least 1500 cells occurs, pro- 
viding that maximal amounts of food have been taken. If smaller 
amounts have been given, an increase of 1000 cells is sufficient to 
establish its existence, provided that the same result is observed on 
repeated examination. 

Leueocytosis of Pregnancy and Parturition. — The hyperleucocy- 
tosis which is observed in pregnancy is particularly marked during 
the last five months, and appears to occur quite constantly in primi- 
para, while in multipara exceptions are common. In an analysis 
of 55 cases Hubbard and White 3 obtained positive results in 44 — 
i. e. y in 80 per cent. — most marked and constant in young primipara. 
Eieder in an analysis of 31 cases noted a hyperleucocytosis in 20, 
all the negative cases being multipara. In a series of 17 multipara 
an increased number of leucocytes was noted in only 7. In Boeder's 
series the number of leucocytes varied between 10,000 and 16,000, 
with an average of 13,000. This represents the usual increase, but 
at times much larger numbers may be observed ; Cabot thus reports 
3 cases with a leueocytosis of from 25,000 to 37,000. 

During actual labor there is an increase of the leucocytes over and 
above the numbers previously observed in pregnancy ; 30,000 cells 
may then be noted. The highest numbers are met with in severe 
and protracted cases, especially after rupture of the waters. This 
form of hyperleucocytosis subsides after the expulsion of the child, 
and at the end of the first or second week normal values are again 
reached, though the gradual decline may be interrupted by a tem- 
porary increase now and then, referable to various minor disturbances 
during the puerperal state. 

As in the case of the digestive leueocytosis, the hyperleucocytosis 
of pregnancy and the puerperal state is brought about by an increase 

1 E. Muller, Zeit. f. Heilk., 1890, p. 213. 

2 Eieder, Beit. z. Kenntniss. d. Leukocytose, 1892. 

3 Hubbard and White, Jour, of Exper. Med., 1898, p. 639. 



PLATE VII. 







! h 



:: V 






-O 





11" 



^ 




m^\ O) 



) 



■*&$&& 










pyrtuniiltftc. 

The Blood of Pernicious Anaemia. 

Note (a) the variations in the size and form of the red corpuscles ; (5) the existence of polychromato- 
philic (i) a and granular b (2) degeneration in some of the red corpuscles ; (c) thepresence of nucleated 
red corpuscles, both of the normoblastic (3) and megaloblastic type (4); (d) the presence of free nuclei 
(5), derived from nucleated red corpuscles. (Bausch and Lomb, Eye-piece 1 inch, objective i-i2th.) 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 89 

both of the polynuclear neutrophiles and the lymphocytes, while the 
eosinophiles remain passive. 

Leucocytosis following Baths, Muscular Exercise, etc. — The in- 
crease of the leucocytes following cold baths may, according to 
Thayer, amount to nearly 300 per cent. 1 In 20 cases of typhoid 
fever he found 7724 leucocytes on an average before, and 13,170 
after the usual Brand bath. In his own person, while in health, the 
leucocytes on one occasion numbered 3250 before the bath, while 
twenty minutes later they had increased to 12,500. Such an 
increase is, however, only observed after a bath of moderate dura- 
tion, while a prolonged cold bath diminishes the number. Hot 
baths have exactly the opposite effect, viz., those of short duration 
produce a decrease, those of long duration an increase. 

Violent muscular exercise, as well as massage, produces a tempo- 
rary hyperleucocytosis. 

Pathological Hyperleucocytosis. — 1. TJie Hyperleucocytosis of the 
Acute Infections. — In the acute infectious diseases hyperleucocytosis 
referable to an increase of the polynuclear neutrophiles is the rule. It 
is thus seen in pneumonia, erysipelas, diphtheria, scarlatina, the various 
septic conditions, parotitis, acute articular rheumatism, etc. Typhoid 
fever and measles represent notable exceptions if we disregard the 
very earliest stage in the development of the disease, when an acute 
hyperleucocytosis may also be observed (see page 96). 

Generally speaking, the increase in the number of the leucocytes 
in the acute infectious diseases is directly proportionate to the 
intensity of the infection and the power of resistance on the part of 
the individual. Where this is particularly feeble or the virulence 
of the infection is especially intense, an absolute increase of the total 
number of the leucocytes may not take place, although a relative 
increase of the polynuclear neutrophilic elements will probably 
always be observed. A recognition of this fact is of importance 
from the standpoint both of prognosis and of diagnosis, and serves to 
illustrate the special value of the differential count. 

In 'pneumonia the increase in the number of the leucocytes is 
usually marked. On an average it amounts to about 24,000 cells 
above the normal (Cabot). The hyperleucocytosis sets in quite early 
— within a few hours following the initial chill — and persists until 
the time of the crisis, when it rapidly disappears ; the decrease may 
indeed precede the critical fall of the temperature. When the disease 
terminates by lysis the return to the normal is more gradual. A 
pseudocrisis is not accompanied by a fall in the number of the 
leucocytes. When resolution is delayed or complications occur, the 
hyperleucocytosis persists. Excepting very mild cases the prognosis 
is especially grave when a well-marked hyperleucocytosis does not 

1 Thayer, Johns Hopkins Hosp. Bull., April, 1893. 



90 THE BLOOD. 

occur. Sears and Larrabee 1 found the mortality much greater when 
the leucocytes numbered less than 10,000 than when they were more 
numerous ; and according to Loper, a progressive increase of the neu- 
trophiles beyond 90 to 95 per cent, may be regarded as an unfavor- 
able symptom irrespective of their total number. Absence of hyper- 
leucocytosis, excepting in very mild cases, will usually warrant a fatal 
prognosis ; exceptions, however, occur, and it is well in any case to 
base prognostic conclusions not upon a single count, but upon the 
result of repeated examinations. It is not uncommon to meet with 
considerable fluctuations in the leucocyte count in the course of the 
disease. Associated with the increase of the polynuclear neutro- 
philes in pneumonia there is a relative diminution of the lympho- 
cytes. The eosinophiles at the height of the disease are greatly 
diminished ; they may indeed be absent. Their return may occur 
before the beginning of the crisis and may be viewed as a favorable 
symptom. 

In bronchopneumonia the total increase of the leucocytes is not so 
extensive as in the acute croupous form. 

In erysipelas, as in pneumonia, the hyperleucocytosis is generally 
proportionate to the intensity of the morbid process and also termi- 
nates by crisis. The increase of the leucocytes beyond normal may 
amount to 15,000 (Rieder) ; in many cases, however, the total 
number does not rise much beyond the upper limit of the normal. 
At the height of the disease the eosinophiles are much diminished or 
absent. 

In diphtheria a well-marked increase is the rule. Generally the 
count does not exceed 25,000 to 30,000, but in fatal cases it is com- 
mon to meet with larger numbers. Ewing 2 speaks of one case with 
lymphocytosis in which the count was 72,000, and cites a peculiar 
instance reported by FelsenthaP marked by hemorrhagic eruption in 
which 148,000 were counted. As Ewing suggests, this was probably 
an agonal hyperleucocytosis. As a rule, from 25,000 to 50,000 cells 
are met with in fatal cases. In children the general increase of the 
leucocytes is frequently associated with a relative lymphocytosis. 
The eosinophiles are diminished in number and may indeed be 
absent. It is interesting to note that excepting a temporary dimi- 
nution immediately following the injection, the leucocytosis is in no 
wise influenced by the antitoxin treatment. Besredka, 4 however, 
states that the grade of the polynuclear neutrophilic hyperleucocytosis 
after the administration of the serum indicates the prognosis. Thus, 
if one or two days after the injection the percentage of the neutro- 
philes is 60 or more, the prognosis is good ; with a higher tempera- 

1 Sears and Larrabee, Med. and Surg. Eep. of the Boston City Hosp., 1901, 12th 
series, Dec. 1st. 

2 Ewing, On the Blood, loc. cit. 

3 Felsenthal, Arch. f. Kinderheilk., vol. xv. p. 78. 

4 Besredka, Annal. de l'lnst. Pasteur, 1898, vol. xii. 5, p. 305. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 91 

ture and 50 per cent, it is bad, and with a lower percentage than 
60 the disease is fatal. The exanthem which occasionally follows 
the injection of antidiphtheritic serum is accompanied by a poly- 
nuclear neutrophilic hyperleucocytosis. 

In tonsillitis there is an increase of the leucocytes of approximately 
the same intensity as in diphtheria, with a similar diminution in the 
number of the eosinophiles. 

In septic conditions, in general, hyperleucocytosis is of constant 
occurrence at some stage of the disease, unless the infection is very 
mild or very severe. Even in those cases in which the absolute 
increase of the leucocytes is not especially marked, or, as in certain 
very virulent cases is absent altogether, the neutrophils are relatively 
increased and the eosinophiles coincidently very much diminished or 
absent altogether. Osteomyelitis forms the only apparent exception 
to this rule in so far as the eosinophiles are concerned. 

Especially important is the study of the leucocytosis in appendi- 
citis. I here quote from Bloodgood's paper on the value of blood 
examination in surgical diagnosis : * 

Observed within forty-eight hours the number of white blood-cells 
is in the majority of instances of great value in pointing to the extent 
of the inflammatory condition of and about the appendix. Cases 
of recurrent appendicitis or of appendicitis suffering from the first 
attack, first observed practically at the end of the attack when the 
clinical symptoms are subsiding, rarely show an increase in the 
white cells. In a few instances, first observed within forty-eight 
hours after the beginning of the attack, but when the symptoms 
are subsiding, there have been a few leucocyte-counts of 15,000, 
which have fallen rapidly within a few hours to 10,000 and 7000. 
In the cases admitted within forty-eight hours with acute symptoms, 
if on account of the clinical picture operation has been delayed, a 
falling leucocytosis has always been observed. These patients have 
recovered, and at a later operation the appendix was found to be 
the seat of a diffuse inflammation, but there was no evidence of 
pus outside the appendix. In one case admitted sixteen hours after 
the beginning of the attack the leucocytes fell in ten hours from 
17,000 to 13,000, and in twenty-four hours to 11,000, associated with 
disappearance of the symptoms. With one exception, the highest 
first leucocyte-count in this group has been 17,000, falling in a few 
hours to 12,000, 9000, or even lower. A patient admitted twenty 
hours after the beginning of the acute attack had a leucocytosis of 
22,000 ; the clinical symptoms, however, were not very marked. 
The patient was observed eight hours ; during this period the leuco- 
cytes fell to 16,000 and the local symptoms practically disappeared. 
Within the succeeding twenty-four hours the leucocytes were 11,000, 

1 Bloodgood, "Blood Examination as an Aid to Surgical Diagnosis/' Am. Med., 
1901, p. 306, 



92 THE BLOOD. 

then 8000, 7000, and 6000. Although this patient with a leucocytosis 
of 22,000 at the end of twenty hours, recovered, and there is 
every reason to believe that the inflammatory condition about the 
appendix subsided, nevertheless it is an exception to the general rule, 
and it would be safer, I believe, to operate in those cases of acute 
appendicitis observed within the first forty-eight hours with a leuco- 
cytosis of 20,000. 

In acute diffuse appendicitis with operation and recovery the highest 
count observed was 25,000 thirty-six hours after the beginning of 
the attack. At operation in this case intense inflammation and a 
large amount of exudate were found about the appendix. 

In gangrenous appendicitis with operation and recovery the leuco- 
cytosis is higher (25,000—35,000) and rises more rapidly. As Blood- 
good says, the study of the leucocytosis is here of the greatest 
importance in the early recognition of a grave inflammatory condi- 
tion of the appendix, which without doubt would lead to general 
peritonitis and death unless early operation be instituted. 

A very high leucocytosis within forty-eight hours after the 
beginning of the attack is suggestive, but not at all positive, of 
beginning peritonitis. The leucocyte-count, however, does not seem 
to help in such cases with regard to prognosis. After the second 
day in cases in which the peritonitis has been present longer Blood- 
good never has observed recovery with a low leucocyte-count. 
If the leucocytosis remains still high at this period, however, the 
prognosis seems better for ultimate recovery after operation. 

In intestinal obstruction an increase of the leucocytes associated 
even with very slight symptoms is of the highest importance in the 
early recognition of the lesion. Bloodgood states that in a large 
group of cases the leucocyte-count may rise to 20,000 within 
twelve hours after the beginning of the obstruction. Within the 
first twelve to twenty -four hours a few observations would demonstrate 
that if the leucocyte-count rise above 25,000 or 30,000, the proba- 
bilities are that one will find gangrene of the obstructed loops or 
beginning peritonitis. If observed on the second or third day 
after the beginning of the symptoms, it is difficult to make a differ- 
ential diagnosis with regard to gangrene or peritonitis. After the 
third day, in cases in which there is no gangrene, and no peritonitis, 
or in which the auto-intoxication is not yet very grave, the 
leucocytes still remain high — 15,000-23,000 — according to the de- 
gree of obstruction : complete, higher ; partial, lower. In the presence 
of gangrene-peritonitis or grave auto-infection, the leucocytes begin 
to fall. If the patient is admitted after the third or fourth day, 
with a history of intestinal obstruction, and still has a high leuco- 
cyte-count, the prognosis is good for operation. If the count is 
low, and especially if it is below 10,000, the probabilities are that 
on operation extensive gangrene-peritonitis will be found; or the 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 93 

patient will be so depressed by auto-intoxication that reaction does 
not follow relief of the obstruction. 

In scarlatina hyperleucocytosis is a constant feature of the disease. 1 
It usually begins two or three days before the appearance of the 
rash ; sometimes even as early as the sixth day. The acme is 
reached on the second or third day ; on the fourth medium values 
are found. Then the decrease usually begins, although this is some- 
times delayed until the eighth or ninth day ; normal values are not 
reached until the end of the second or the beginning of the third 
week. In light cases the leucocytosis amounts to from 10,000 to 
20,000 cells, in cases of moderate severity 20,000 to 30,000 are 
average figures, while in fatal cases 40,000 are common values. 
The hyperleucocytosis is scarcely influenced by the height of the 
temperature, the angina, the rash, desquamation, or complications, 
excepting that in the latter case its duration is influenced by the 
nature of the pathological process. The hyperleucocytosis is due to 
a large increase of the polynuclear neutrophiles, which may represent 
94 per cent, of all leucocytes. The lymphocytes are proportionately 
diminished unless glandular complications occur, when they may 
reach maximum normal values. The eosinophiles in light and 
moderately severe cases are at first normal or subnormal, they then 
gradually increase and reach maximum values (8-15 per cent.) in 
the second or third week, after which they return to normal. In 
severe cases they diminish to zero (see also page 101). 

In acute articular rheumatism the degree of hyperleucocytosis is 
proportionate to the severity of the attack. In McCrae's 2 analysis 
of 83 cases the average count was 11,776; in 29 it was below 
10,000. Taking the average of the remaining 54 cases we have 
14,260. In 17 the count was over 15,000 and in 4 over 20,000; 
the highest figure was 38,000. It is noteworthy that hyperleuco- 
cytosis was noted in all cases of complicating pericarditis in which a 
count was made, but that normal values were obtained in many cases 
of undoubted endocarditis. In pericarditis 15,000 to 19,000 were 
average values ; 35,000 was the highest count noted. Generally 
speaking, when the number of leucocytes in acute articular rheu- 
matism rises to 20,000 or higher, pericarditis or pneumonia may be 
suspected (Turk, Ewing). When the total increase of the leuco- 
cytes is only slight, the percentage values are not especially disturbed, 
but with a marked hyperleucocytosis the polynuclear neutrophiles 
are materially increased. The eosinophiles are commonly absent in 
the early stages of the disease, while later they are always present 
in moderate numbers, and after defervescence they are usually 
increased. 

1 Van der Berg, Arch. f. Kinderheilk., vol. xxv. p. 321. Mackie, Lancet, Aug. 24, 
1901. Keckzeh, Zeit. f. klin. Med., 1902, vol. xliv. p. 201 (full literature). 

2 McCrae, Jour. Am. Med. Assoc, 1903, vol. xl. p. 210. 



94 THE BLOOD. 

In tubercular disease hyperleucocytosis is observed only when 
secondary infection with pus organisms has taken place, while in 
pure cases the number remains normal. As the conditions for a 
secondary infection are more favorable in some parts of the body 
than in others, such as the lungs and kidneys, hyperleucocytosis is 
commonly present when these parts are involved. In the third 
stage of pulmonary tuberculosis there is usually a leucocytosis of 
from 15,000 to 20,000, which is referable to a well-marked increase 
of the polynuclear neutrophiles, while the eosinophiles are diminished. 
In the second stage, owing to a concentration of the blood no doubt, 
values ranging between 8000 and 10,000 are common, while in the 
first stage normal values are found. 1 In tubercular peritonitis the 
leucocytosis is variable. In 36 cases of 46 analyzed by Shattuck 
the number was below 10,000 ; where it is higher pus may or may 
not be present. In tubercular meningitis there is as a rule no 
increase in the number of the leucocytes ; but in a few instances 
counts between 14,000 and 34,000 have been reported. When 
hyperleucocytosis does occur, it may be due to a complicating ter- 
minal pneumonia. 

In non-tubercular meningitis hyperleucocytosis is well marked ; it 
appears early in the disease and persists until death. 

In smallpox a hyperleucocytosis is observed only in the severer 
cases and when pustulation takes place. In the milder forms no 
increase occurs. 

In Malta fever a marked increase of the polynuclear neutrophiles 
may occur just before the onset of the fever ; later there is absence 
of hyperleucocytosis (Bruce). In a case observed in the United 
States by Musser and Sailer 11,564 leucocytes were counted, all 
varieties being present in normal proportion. 2 

In bubonic plague a moderate increase of the leucocytes is the 
rule ; a few instances have been reported in which over 1 00,000 
cells were counted, the increase being largely due to the neutrophiles. 

In uncomplicated cases of typhoid fever , as I have indicated, the 
leucocytes are diminished except during the first days, when there 
may be a leucocytosis of 3000-5000 beyond the normal, referable 
to an increase of polynuclear neutrophiles. After this, however, 
the leucocytes diminish and a relative lymphocytosis gradually comes 
to the foreground (see especially page 96). 

In uncomplicated measles there is in the beginning a moderate 
relative increase of the polynuclear neutrophiles, 76-82 per cent. ; 
but this is not associated with an absolute increase of the leucocytes, 
but with a decrease. Later there is a relative decrease of the neutro- 
philes to 50-60 per cent., while the absolute number is increased. 

According to Wilson and Chowning, a hyperleucocytosis of about 

1 Appelbaum, loc. cit., p. 61. 

2 Musser and Sailer, Phila. Med. Jour., 1898, p. 1408, and 1899, p. 89. 



« 

MICROSCOPICAL EXAMINATION OF THE BLOOD. 95 

12,000 is usual in cases of the so-called spotted fever of the Rocky 
Mountains. 1 

2. Ancemic Hyperleucocytosis. — Hyperleucocytosis referable to an 
increase of the polynuclear neutrophiles is observed in various forms 
of acute and chronic anaemia. It is especially marked after hemor- 
rhages, when the number of leucocytes may increase to 30,000 and 
even more. Generally speaking, the degree of increase is here 
proportionate to the amount of blood lost and the recuperative power 
of the individual. In the human being Rieder noted a leucocytosis 
of 15,000 after a pulmonary hemorrhage ; 32,600 after a hemorrhage 
due to uteric cancer, and 26,500 after a hemorrhage referable to 
gastric ulcer. 

If we except the myelogenous type of leukaemia, in which an 
absolute increase of the polynuclear neutrophiles is associated with 
a relative decrease, hyperleucocytosis is not met with in uncomplicated 
cases of the primary anaemias. In the secondary forms, however, 
it is quite common, though usually of moderate degree. 

3. Cachectic Hyperleucocytosis. — A cachectic hyperleucocytosis has 
been described in connection with malignant disease, phthisis, etc. 
Ewing states that in the majority of cases of tertiary syphilis, 
tuberculosis, and nephritis, in a large proportion of carcinoma cases 
and in a rather smaller proportion of sarcomas the cachexia is 
unaccompanied by hyperleucocytosis unless there is distinct local 
inflammation, necrosis, or hemorrhage. He suggests that the exist- 
ence of a marked hyperleucocytosis in the course of a cachexia 
should lead to a search for one of these complications. 

4. Ante-mortem Hyperleucocytosis.- — An ante-mortem hyperleuco- 
cytosis has been described by Litten 2 and others in moribund indi- 
viduals, in which no increase in the leucocytes had previously 
occurred. 

5. Hyperleucocytosis referable to Drugs. — Hyperleucocytosis refer- 
able to an increase of the polynuclear neutrophiles has been observed 
in cases of poisoning with potassium chlorate, arsenions hydride, 
and illuminating gas. It follows the administration of atropine, 
quinine, the salicylates, thyroid extract, tuberculin, and the infusion 
of salt solution. It is noted after prolonged anaesthesia with chloro- 
form and ether, when an increase of 5000-10,000 cells is quite 
common. This increase occurs after from six to forty-eight hours 
following the operation, and persists for only a few hours. A post- 
operative increase of 10,000 or more beyond the normal value of the 
individual and sustained for more than a few hours, should be 
looked upon with suspicion. 3 

6. Hyperleucocytosis of Thermic Fever. — In thermic fever a high 

1 Wilson and Chowning, Jour. Am. Med. Assoc, July 19, 1902, p. 131. 

2 Litten, Berl. klin. Woch., 1877, No. 51. 

3 Da Costa and Kalteyer, Am. Med., 1901, p. 306. 



96 THE BLOOD. 

leucocyte count is apparently the rule, but there is considerable 
irregularity in the time and duration of the rise. Lewis and 
Packard 1 report that in some of their cases a leucocytosis of from 
12,000 to 13,000 was noted on admission. In most of the cases in 
which there was a primary rise this was followed by a fall and then 
a second increase in their number. 

In addition to these various forms of hyperleucocytosis an increase 
of the neutrophiles is further observed under conditions which do 
not as yet permit of an appropriate classification ; in some cases 
no doubt the hyperleucocytosis is of toxic origin ; in others it may 
be referable to an abnormal distribution of the cells ; in still others 
to a coexistent anaemia, etc. Such conditions are rickets, gout, acute 
yellow atrophy, advanced hepatic cirrhosis (especially when associated 
with jaundice), acute gastro-intestinal disorders, acute and chronic 
nephritis, hydronephrosis, etc. 

Polynuclear Neutrophilic Hypoleucocytosis (Leukopenia). — 

A diminution in the total number of the leucocytes is observed in 
only a comparatively small number of diseases, and is practically 
always referable to a decrease in the number of the polynuclear 
neutrophiles. It is notably observed in typhoid fever, measles, 
influenza, in certain anaemic conditions, etc. 

In typhoid, fever 2 hypoleucocytosis is so constantly seen that we 
can formulate the general rule that whenever an increase in the num- 
ber of the leucocytes is observed in a case of suspected typhoid fever it 
is more than probable that some complication exists or that the diagnosis 
is wrong. Exceptions to this rule are rare. In the very earliest 
days of the disease, possibly owing to a concentration of the blood, 
the result of starvation and diarrhoea, higher counts are sometimes 
observed, but as the disease progresses the number soon diminishes, 
and in the later stages of the disease is practically always markedly 
below the normal. Not uncommonly they are less than 2000, and 
in some instances the number may indeed fall below 1000. The 
qualitative changes are especially important ; and according to 
Nageli, characteristic of the different stages of the disease. At first, 
while the temperature is steadily rising there is a neutrophilic hyper- 
leucocytosis of moderate degree ; this is associated with a moderate 
decrease of the lymphocytes, while the eosinophiles disappear. Then 
the neutrophiles diminish and the period of the hypoleucocytosis 
properly speaking commences. During this stage, viz., the stage of 
continued fever, the neutrophiles usually number from 3000 to 4000, 
as compared with 5000 to 6000 during the second half of the first 

1 Lewis and Packard, Trans. Assoc. Am. Phys., 1902, p. 409. 

2 Nageli, Deutsch. Archiv, lxvii., Parts 3 and 4. Kolner, Ibid., lx. p. 221. Thayer, 
Johns Hopkins Hosp. Bull., 1901, vol. iii. p. 500; and Studies in Typhoid Fever, Johns 
Hopkins Press, 1901, p. 487. 



PLATE VIII. 













• 



C 



^ 



w v 



w 




I -") 



<r* 



n 







/ **?* N 



^(p. s 






2 \|> : i# 2 







Mjjthmi'df. fpf. 



The Blood of Lymphatic Leukaemia. 



Note the large increase of the lymphocytes Two of th#» t-^ 

oe^uo,. « ; ta . fcw il ^^cs: ,t«^ssls r.?r^r nur 

ph.hc leucocyte .s seen with scattered grounds. (Bausch and Lotnb 
Eye-piece i inch, objective i-i2th.) 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 97 

week. The lymphocytes are now also diminished, but tend to rise 
toward the end of this period ; the eosinophiles are absent. During 
the third stage (remission) the neutrophiles decrease still further — 
1500—2500 — while the lymphocytes increase and a few eosinophiles 
appear. In the fourth stage (defervescence) the neutrophiles reach 
their minimum, 900 in severe cases, while the lymphocytes are 
relatively much increased and the eosinophiles gradually return to 
normal. The reascent of the neutrophiles then occurs very slowly, 
while coincidently there is a lymphocytosis which is especially marked 
in children and continues far into convalescence. Normal values 
are sometimes not reached until after a couple of months. 

In the event of a relapse occurring during an afebrile period there 
is a distinct neutrophilic hyperleucocytosis, the actual number depend- 
ing upon the preceding counts, to which from 3500 to 5000 neutro- 
philes are added ; at the same time the eosinophiles disappear. Should 
a relapse occur in the third stage of the disease, then the eosinophiles, 
which have just begun to reappear, disappear abruptly. 

Favorable indications in cases of typhoid fever are an increase of 
the eosinophiles at the height of the disease ; reappearance of the 
eosinophiles, indicating arrival at the third or fourth stage ; an 
increase of the lymphocytes, which appears to begin only at a time 
when the severest part of the intoxication is over ; not too great a 
decrease of the neutrophiles in the absence of complications. Un- 
favorable indications are : a marked decrease of all leucocytes, and 
especially of the lymphocytes ; absence of hyperleucocytosis and a 
farther decrease of the neutrophiles in the event of complications, 
which per se would call forth a hyperleucocytosis. 

In the event of complications the total number of the leucocytes 
frequently does not exceed the upper limit of the normal ; but in 
such cases a differential count will reveal a relative increase of the 
neutrophiles. 

In cases of perforation there is frequently an increase in the total 
number of the leucocytes, which may, however, be quite transitory 
and escape observation unless an early examination is made and 
previous counts are available ; for later, when peritonitis is general, 
the leucocytes are usually found diminished ; in some instances, 
however, there is no increase at the onset. 

In one of Cabot's cases the count before operation was 8300, and 
immediately afterward 24,000. Finney reports a case with 6500 
before and 10,600 after. In one of dishing' s cases there was 
an early recognized hyperleucocytosis which appeared before any 
sign of general peritonitis had developed; 8400 before and 16,000 
after. In this patient it was interesting to note that following 
the operation the leucocytes fell to 4000 ; but immediately fol- 
lowing the development of obstruction, due to kinking of the 
bowel, the leucocytes increased to 13,000 and later to 20,000, to 

7 



98 THE BLOOD. 

fall again following the removal of the obstruction. In a second 
case operated by Cashing there was a persisting hyperleucocytosis, 
associated with abdominal pain and tenderness, at one time reaching 
15,200. Upon the development of general peritonitis the count 
showed only 4300. Cabot remarks, " steadily increasing leukocyto- 
sis is always a bad sign, and should never be disregarded, even when 
other bad symptoms are absent," to which Cushing adds, " a decreas- 
ing leucocytosis may be a much worse sign " (Finney 1 ). 

Measles 2, is the second notable exception to the general rule that 
the acute infections are associated with a poly nuclear neutrophilic 
hyperleucocytosis. But it is interesting to note that here also the 
hypoleucocytosis is preceded by a pre-eruptive hyperleucocytosis, 
which commences at the beginning of the period of incubation, then 
increases rapidly and reaches its maximum about the sixth day 
before the appearance of the eruption. After this it diminishes, and 
at the appearance of the exanthem and during its course the occur- 
rence of an increased number of leucocytes indicates some complica- 
tion. The hypoleucocytosis affects the polynuclear neutrophiles both 
absolutely and relatively, while the lymphocytes are relatively at 
least increased. The eosinophiles disappear. The hypoleucocytosis 
generally reaches its maximum on the second day of the eruptive 
stage, when the number of leucocytes is reduced to about one-half. 
After this they increase again more or less rapidly and reach the 
normal from one to five days after the disappearance of the rash, 
unless some complication should supervene. Numerous eosinophiles 
then appear together with an absolute and relative increase of the 
polynuclear neutrophiles. 3 

Urticaria, syphilitic roseola, scarlatina, and the exanthem which 
may follow antitoxin treatment are not associated with hypoleuco- 
cytosis. 

In uncomplicated cases of tuberculosis there is usually no increase 
of the leucocytes ; Avhen it does occur it is generally referable to 
suppurating cavities, recent hemorrhages, and resulting anaemia, or 
to advancing pneumonia. The increase which occurs under such 
conditions is moderate and does not often exceed 15,000 cells. 
Ewing states that he has seen both lungs consolidated and riddled 
with small cavities in a case lasting but five weeks, and yet the 
leucocytes were never found above 12,000. He suggests that the 
absence of leucocytosis in such cases of acute phthisis which resemble 
pneumonia may often be of value in diagnosis. Unfortunately there 
is no large series of examinations available from which to decide the 
relative value of the morphological examinations of the blood in the 
differential diagnosis between acute miliary tuberculosis and typhoid 

1 J. M. T. Finney, Surgical Treatment of Perforating Typhoid Ulcer. Studies in 
Typhoid Fever. Johns Hopkins Press, 1901, p. 170. 

2 Eeckzeh, Zeit. f. klin. Med., vol. xlv. p. 107 (full literature), 
? Renaud, These de Lausanne, 1900. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 99 

fever. According to Cabot and Warthin, a subnormal number of 
leucocytes may also be observed in acute miliary tuberculosis, while 
Kolner 1 thinks the leucocyte count important in distinguishing 
between the two diseases. 

In uncomplicated cases of influenza the total number of the leuco- 
cytes is commonly diminished ; it may, however, be normal. In my 
experience there is usually a slight relative increase of the neutro- 
philes with diminution of the eosinophiles. When an absolute increase 
of the leucocytes occurs, some complication probably always exists. 

In severe cases of anosmia the occurrence of hypoleucocytosis is 
always a grave symptom, as it indicates an inability on the part of 
the bone-marrow to produce a sufficient number of blood-corpuscles. 
Ehrlich supposes that in such cases the fatty marrow of the long 
bones is not transformed into red marrow, and he cites two cases in 
which the correctness of this supposition was demonstrated at the 
post-mortem table. A hypoleucocytosis of this order was observed 
by Decastello and Hofbauer 2 in five cases of pernicious anaemia, in 
four of chlorosis, in two of post-hemorrhagic anaemia, in two of liver 
abscess, one of phthisis florida, one of sepsis with severe anaemia, in 
three severe anaemias of unknown origin, in two cases of pseudo- 
leukaemia, and two of splenic anaemia. 

In splenic anaemia 3 hypoleucocytosis appears to be a feature of 
the disease at some period in its course and is at times most marked. 
Osier mentions a case of Vickery's in which only 650 to 700 leuco- 
cytes were counted pro cbmm., and one of Peabody's with 800 cells. 
The average count in the series collected by Osier was 3850. 
Immediately after a profuse hemorrhage or in a terminal affair there 
may be a hyperleucocytosis. 

In pernicious anaemia hypoleucocytosis is also the rule, and at 
times remarkably low counts are obtained. Strauss and Rohnstein 4 
cite two cases with 400 and 328 cells, respectively. As a rule, 
however, the diminution is more moderate, and the general average 
not much below the minimum normal. 

While the hypoleucocytosis in the diseases mentioned is rarely 
extreme, most extraordinary instances of leukopenia are at times 
encountered. Ehrlich 5 thus cites the case of a well-built young 
man in whom brief epileptiform seizures occurred, and in one of 
which the patient died. The post-mortem examination was entirely 
negative. During the three days of observation preceding death two 
examinations of the blood were made. On the first day not a single 
leucocyte could be demonstrated in ten blood-films, and on the 
second day but one was found in the same number of specimens. 

1 Loc. cit., p. 96. 

2 Decastello and Hofbauer, Zeit. f. klin. Med., vol. xxxix. p. 488. 

3 Osier, Am. Jour. Med. Sci., 1902, vol. cxxiv. p. 751. 
4 Strauss u. Rohnstein, loc. cit. 

5 Ehrlich^ Die Ansemie, loc cit, 
L. of O. 



100 THE BLOOD. 

Of drugs, atropine, camphoric acid, tannic acid, picrotoxin, agaricin, 
menthol, sulphonal, and several other antihydrotics, cause a marked 
decrease of the leucocytes. 1 

Polynuclear Eosinophilic Hyperleucocytosis (Eosinophilia), 

— A 'physiological increase of the eosinophils beyond the maximum 
observed in adults is seen in young children. According to Zap- 
pert, the relative numbers may here vary between 0.67 and 11 per 
cent., and Muller and Kieder even speak of 21 per cent. In older 
children, however, normal adult values prevail, and it is then legiti- 
mate to consider an increase beyond these figures as abnormal. 

It is stated by some that there is a physiological increase of the 
eosinophils during the menstrual period and following coitus. This 
is inconstant, however, and rarely marked. 

Eosinophilia is thus essentially a, pathological phenomenon. It 
occurs under the most diverse conditions, as in the myelogenous type 
of leukaemia, in bronchial asthma, in various skin affections, the 
helminthiases, gonorrhoea, osteomyelitis, following the injection of 
tuberculin, etc. 

In myelogenous leukcemia (Ehrlich) an absolute increase in the 
number of eosinophiles is one of the most constant symptoms of the 
disease. Ehrlich indeed has taught that this increase occurs in all 
cases and must be demonstrable to warrant the diagnosis. In view 
of recent advances in our knowledge of the pathology of the disease, 
however, this idea can no longer be upheld, as it has been shown 
that all forms of leukaemia are or at least may be of myelogenous 
origin. 2 Cases have been recorded in which the blood picture was 
essentially that of the orthodox lymphatic variety, but in which 
post-mortem examination showed a total absence of involvement 
of the lymph-glands, while the bone-marrow was extensively dis- 
eased. In these cases there was no increase in the total number of 
the eosinophiles. But it seems that even in those cases in which the 
blood picture is essentially that of a myelaemia the usual increase in 
the number of the eosinophiles may be lacking. I have thus 
reported an instance in which these cells were not only not increased, 
but were practically absent. 3 Such cases, however, are exceedingly 
rare, and it may still be regarded as the rule that in those cases 
of leukaemia in which extensive myelocytosis exists the eosinophiles 
are absolutely if not relatively increased. With septic complications 
occurring in the course of the leukaemias the eosinophilic leucocytes 
are materially diminished, and in some cases they may be absent 
altogether. Exceptions, however, occur, and Ehrlich cites a case in 
which the absolute number of eosinophiles was still between 1400 

iBohland, Centralbl. f. inn. Med., 1899, No. 15. 
2 Pappenheini, Zeit. f. klin. Med., vol. xlvii. p. 216. 
3 C. E. Simon, Am. Jour. Med. Sci., June, 1903. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 101 

and 1500 pro cbmm., although the percentage had diminished from 
3.5 to 0.43. 

In bronchial asthma an increase of the eosinophils is observed 
quite constantly about the time of the paroxysm, and may amount 
to from 10 to 53.6 per cent. 1 Its occurrence is of value in differ- 
ential diagnosis as renal and cardiac Asthma are not associated with 
eosinophilia. Between attacks normal numbers are found (v. Noor- 
den, Swerschewski). 

In scarlatina 2 an increased number of eosinophils is quite con- 
stantly observed at some time in the course of the disease. As the 
result of an analysis of 167 cases Bowie finds that at the onset of 
the fever they are diminished. In simple favorable cases they then 
increase rapidly until the height of the disease is passed, when they 
diminish again, and finally reach the normal some time after the 
general hyperleucocytosis has disappeared, viz., when the poison has 
all been eliminated. The more severe the case the longer are the 
eosinophiles subnormal before they rise again ; in fatal cases they 
never rise, but rapidly decrease to zero. Bowie thinks that the 
curve of the eosinophiles is of value from a prognostic standpoint. 
If they are normal or subnormal after the first day or two, the 
case will in all probability be a severe one. In Reekzeh's series 
the highest percentage was 12.5, and the largest total number 
1350. 

In measles an increase of the eosinophiles does not occur. 

In many skin diseases eosinophilia may also occur, as in pem- 
phigus, prurigo, psoriasis, in Duh ring's hydroa gravis, in urticaria, 
chronic eczema, etc. Generally speaking, the degree of increase in 
skin diseases is proportionate to the amount of tissue involved. In 
urticaria and pemphigus more particularly the increase may be 
very marked ; in one case of urticaria 60 per cent, were noted, and 
in a case of pemphigus the absolute number of eosinophiles was 4800 
(as compared with 250, the highest normal value). In some cases 
of leprosy percentages varying between 8.48 and 61 have been 
obtained, while in others, notably in the nervous form, normal 
values are found. In a case of epidermolysis bullosa hereditaria 
Brown met with 9.7 per cent, of eosinophiles ; in one of severe 
chronic eczema with 24 per cent. 

Of special interest is the increase of the eosinophiles in the hel- 
minthiases. It may occur in connection with all intestinal parasites, 
including the amoeba. According to Leich ten stern, 3 it is most pro- 
nounced in those cases in which Charcot-Leyden crystals are numerous 
in the feces. The greatest increase has been observed in ankylostomia- 
sis, where 72 per cent, were counted in one case. As a general rule, 

1 Billings, N. Y. Med. Jour., vol. lxv. p. 691. 

2 Zappert, Zeit. f. klin. Med., 1893, p. 292. Keckzeh, Ibid., vol. xlv. (literature). 
Bowie, Jour. Path. u. Bact., 1902, vol. viii. p. 82. 

3 Leichtenstern, Wieu. klin. Rundschau, 1898 ; and Deutsch. med. Woch., 1887-88. 



102 THE BLOOD. 

however, the eosinophilia is not so extensive. Herrick observed one 
case in which the cells numbered 26 per cent., and Kieffer gives 
3—30 per cent, as usual values. In the presence of oxyurides 
Buckler l found 16 per cent. ; 19 per cent, were counted in associa- 
tion with ascarides, and Leichtenstern reports one case of Taenia 
mediocanellata with 34 per cent. It is to be noted, however, that 
eosinophilia is not a constant feature in infections with the common 
taeniae, oxyuris, and ascaris, and that the number of eosinophiles 
may not exceed minimum normal values. In cases of infection 
with the bothriocephalus eosinophilia does not occur (Schaumann). 
In a fatal infection with Balantidium coli Strong and Musgrave 2 
observed a relative increase, and it appears that in amoebic colitis 
also a moderate eosinophilia is not uncommon. 3 

As Brown 4 has shown, a remarkable increase of the eosinophiles 
occurs in trichinosis during the acute stage. In his first 4 cases 
with a total leucocyte count of 35,000, 13J000, 17,000, and 18,000 
the percentage of eosinophiles was 68.2, 42.8, 49, and 48, respec- 
tively. Kerr noted even a higher percentage in one case, viz., 86.6. 
Similar results have been obtained by Thayer, 5 Cabot, 6 Gwyn, 7 
Blumer-Neumann, 8 and others, and it can now be regarded as an 
established fact that the occurrence of eosinophilia is one of the 
most constant and diagnostically important symptoms of the dis- 
ease. That it does not occur invariably, however, is shown by the 
reports of Howard, Da Costa, Drake, and Cutler. 9 A very interest- 
ing case of trichinosis is reported by McCrae, 10 in which the disease 
was complicated by typhoid fever ; the eosinophilia was here never- 
theless well marked. 

In jilariasis also eosinophilia may occur. As the result of his 
study of four cases of the disease in the Philippines Calvert 11 concludes 
that in the early stages hyperleucocytosis with an increase of the 
eosinophiles may be looked for, but that the number of the leucocytes 
in general, as also of the eosinophiles, returns to normal as the disease 
progresses. In one of his cases the percentage increased to 22, but 
varied within twenty-four hours between this point and 8. In a 
case of long standing which I had occasion to examine I found but 
2 per cent, of eosinophiles, with 36 per cent, of lymphocytes. Cal- 
vert, on the other hand, noted no increase of the lymphocytes. A 



1 Buckler, Munch, med. Woch., 1894, Nos. 2 and 3. 

2 Strong and Musgrave, Johns Hopkins Hosp. Bull., 1901. 

3 Amherg, " Amoebic Colitis in Children," Johns Hopkins Hosp. Bull., 1901. 

4 Brown, Jour. Exp. Med., vol. iii. p. 315; and Johns Hopkins Hosp. Bull., 1897. 

5 Thayer, Phila. Med. Jour., vol. i. p. 654. 

6 Cabot, Boston Med. and Surg. Jour., vol. cxxxvii. p. 676. 

7 Centralbl. f. Bakt., vol. xxv. p. 746. 

8 Blumer-Neumann, Am. Jour. Med. Sci., vol. cxix. p. 14. 

9 Cutler, Trans. Assoc. Am. Phys., 1902, p. 356. 

10 McCrae, Am. Jour. Med. Sci., 1902, vol. cxxiv. p. 56. 

11 Calvert, Johns Hopkins Hosp. Bull., 1902, vol. xiii. p. 133. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 103 

relation between the number of embryos and the percentage of the 
different leucocytes does not appear to exist. 

Eosinophilia has further been noted in hydatid disease. Seligmann 
and Dudgeon x thus report a case of hydatid disease of the liver in 
which there was a leucocytosis of 17,111 with 57 per cent, of 
eosinophiles. After operation the leucocytes diminished to 7000 
and the eosinophiles to 1 per cent. A similar case has been reported 
by Bloch 2 with 14.7 per cent, of eosinophiles, and it is noteworthy 
that the cyst at the time was undergoing suppuration. Four weeks 
after operation normal values were obtained. In three other cases 
of hydatid disease reported by Bloch, involving the lung and the 
spleen, there was no increase of the eosinophiles and in one a marked 
lymphocytosis. 

In malaria, the eosinophiles are commonly present in increased 
numbers during the afebrile period, and rarely diminish below the 
minimum normal values even at the time of a paroxysm. Zappert 3 
reports a case of malaria in which on the day following the last 
attack 20.34 per cent. (1486 absolute) were found. 

In malignant disease eosinophilia apparently occurs in only a rela- 
tively small percentage of cases, and when present is usually of 
moderate grade — i. e., not exceeding 7—10 per cent. Occasionally, 
however, the increase is most remarkable. Reinbach thus cites a 
case of lymphosarcoma (malignant lymphoma) of the neck with 
metastases in the bone-marrow, in which 60,000 eosinophilic leuco- 
cytes were counted on one occasion. 

A gonorrheal eosinophilia has been noted by various observers. 
From an analysis of 45 cases which O wings studied in my laboratory 
it appears that with an extension of the inflammatory process to the 
posterior urethra the number of cases increases in which an increased 
percentage of eosinophiles is found in the blood, and in cases of 
prostatitis eosinophilia is the rule. During the first week of the 
disease the blood is apparently always normal. In the second and 
third weeks it is normal in only 33 per cent, of all cases, and after 
two months' duration an increased number is still observed in 40 
per cent. The percentage of the eosinophiles usually does not 
exceed 12 per cent. At times, however, larger numbers are found ; 
Bettmann cites a case of gonorrheal epididymitis with 25 per cent. 
Occasionally the eosinophilia is associated with a neutrophilic hyper- 
leucocytosis ; this is usually of moderate intensity, but may be 
quite marked when the urethritis is complicated by an epididymitis, 
an orchitis, or a cystitis. 

In association with chronic tumors of the spleen and after extirpa- 
tion of the organ eosinophilia has been repeatedly observed. Miiller 

1 Seligmann and Dudgeon, Lancet, June 21, 1902. 

2 Bloch, Deutsch. raed. Woch., No. 29, 1903. 

3 Zappert, Zeit. f. klin. Med., vol. xxiii. p. 227. 



104 THE BLOOD. 

and Rieder 1 report three cases of tumor referable to congenital 
syphilis, hepatic cirrhosis, and neoplasm of the cranial cavity, in 
which 12.3, 7, and 6.5 per cent., respectively, were found. After 
extirpation of the spleen eosinophilia is not immediately observed, 
but develops only after many months and is of moderate grade. 

As I have pointed out, the eosinophilic leucocytes are relatively 
diminished and may disappear altogether in the great majority of 
the acute infectious diseases, with the exception of scarlatina per- 
haps, while hyperleucocytosis referable to the polynuclear neutro- 
philic cells exists. In the post-febrile period, however, the upper 
limit of the normal and even a well-marked eosinophilia are often 
observed. Turk 2 thus found an epicritie eosinophilia of 5.67 per 
cent. (430 absolute) in a case of pneumonia, and after an attack of 
acute articular rheumatism 9.37 per cent. (970 absolute). I have 
recently seen an eosinophilia of 10.5 per cent, after pneumonia. 

An eosinophilia referable to drugs has been described, but has 
attracted little attention. Two cases are reported by v. Noorden, 
who observed an increase of the eosinophiles to 9 per cent. Both 
were cases of chlorosis, and in both the eosinophilia followed the 
internal administration of camphor. Similar observations have been 
made in animals after poisoning with carbon dioxide. 

Following the injection of tuberculin an increase of the eosino- 
philes has been observed in those cases in which a febrile reaction 
had occurred. In one case reported by Grawitz the eosinophilia 
reached its highest point, viz., 41,000, three weeks after the 
injections had been stopped. 

Polynuclear Eosinophilic Hypoleucocytosis (Hypo-eosino- 
philia). — A diminution in the number of the eosinophiles is notably 
observed in the acute infectious diseases which are associated with a 
neutrophilic hyperleucocytosis. The only exception to this rule appar- 
ently is scarlatina ; but here also their number is diminished at the 
onset of the fever, and, as I have stated, in fatal cases they rapidly 
disappear. Aside from the infections which lead to an increase 
of the polynuclear neutrophiles, hypo-eosinophilia also occurs in 
those forms which, like measles and typhoid fever, are associated 
with a decrease of the leucocytes. We may accordingly formulate 
the general rule that a diminution in the number of the eosinophiles 
will be observed at some period in the course of the various acute 
infectious diseases, no matter whether they are associated with a 
general polynuclear hyperleucocytosis or not. The extent to which 
this may go is variable ; in the milder infections the values may be 
but little, if any, below the minimum normal, but in the severer and 
more protracted cases not a single eosinophile may be met with in a 

1 Miiller and Rieder, Deutsch. Archiv, vol. xlviii. p. 105. 
2 Turk, Klinische Blutuntersuchungen, Wien, 1898. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 105 

differential count of a thousand. Whether or not cases occur in 
which they are wholly absent I am not prepared to say. 

Aside from the acute infectious diseases it is uncommon to meet 
with a material diminution of the eosinophiles. It has been observed 
after severe muscular exercise and after castration, and it is com- 
monly noted in lymphatic leukaemia with high lymphocyte counts. 
DaCosta 1 states that he has found a decrease or even an absence 
of eosinophiles in the majority of cases of chlorosis and pernicious 
anaemia. This decrease in pernicious anaemia has also been observed 
by others, 2 and is apparently the rule during the active stage of the 
diseases ; in the interval, however, normal and even supernormal 
values may be obtained. 

The most extensive diminution of eosinophiles which I have per- 
sonally observed occurred very curiously in a case of myelogenous 
leukaemia ; but two eosinophiles were seen during a differentiation 
of many thousand cells. 3 

Lymphocytosis. — According to Ehrlich's conception of lympho- 
cytosis as a passive hyperleucocytosis, an increased number of lympho- 
cytes will be found in the blood whenever an increased circulation 
of lymph occurs in more or less extensive lymphatic districts, the 
lymphocytes being mechanically washed into the blood current. 
But, as I have pointed out, there is evidence to show that the 
lymphocytes also may follow the laws of chemotaxis, and that an 
active lymphocytosis may possibly occur which is quite analogous to 
the hyperleucocytoses referable to the polynuclear granular elements. 4 

Under physiological conditions an increased number of lympho- 
cytes is notably observed in early childhood. Following the tem- 
porary increase of the polynuclear neutrophils which occurs during 
the first twenty-four hours, the lymphocytes rapidly increase in 
number, so that by the twelfth day they represent 45 per cent, of 
all leucocytes (Carstanjen). Gundobin gives 59 per cent, as an 
average value for sucklings as compared with 34.6 per cent, of poly- 
nuclear neutrophiles. In adult life a physiological increase of the 
lymphocytes is notably seen in connection with the increase of the 
polynuclear neutrophiles which occurs during the process of digestion. 

Under pathological conditions lymphocytosis is more common in 
children than in adults, and it is noteworthy that in anaemic and 
poorly developed children the normal ratio of lymphocytes to the 
polynuclear neutrophiles is reached only late. As a general rule the 
increase of the lymphocytes is not excessive and does not raise the 

Clinical Hematology, Blakiston, Phila., 1901. 
2 Strauss u. Eohnstein, loc. cit., p. 31. 

3 Simon, Am. Jour. Med. Sci., June, 1903. 

4 Jolly, " Sur les mouvements amoeboides des globules blancs," etc., Compt. rend, de 
la soc. d. biol., 1898, vol. x. serie v. ; and Wolff, " Giebt es eine aktive Lympbocytose," 
Deutscb. Aerzte-Zeit., 1901, No. 18. 



106 THE BLOOD. 

total leucocyte count much above 30,000 to 40,000. Lymphocytosis 
of this order is notably seen in rickets, in whooping-cough, measles, 
congenital syphilis, in various subacute intestinal disorders of child- 
hood, at times in bronchopneumonia, etc. 

In whooping-cough during the convulsive stage the total number 
of the leucocytes may be increased to four times the normal number ; 
the average in De Amicis and PacchioniV series was 17,943. 
According to these observers, the hyperleucocytosis is demonstrable 
on the first day of the disease ; it reaches its highest point in the 
convulsive stage and persists some time after cessation of the typi- 
cal symptoms. Wanstall 2 in his series of 16 cases, on the other 
hand, finds no evidence of a marked general hyperleucocytosis, and re- 
ports that in some the leucocytes were actually decreased. He could 
demonstrate a well-marked lymphocytosis during the catarrhal stage, 
however, in almost every case, which varied between 40 and 60 per 
cent. Wanstall concludes that an increased percentage of lympho- 
cytes, at least equalling if not exceeding that of the polynuclear 
neutrophiles, is a valuable aid in the diagnosis of whooping-cough 
before the characteristic symptoms of the disease have appeared. 
Exceptions, however, occur, in which the lymphocytosis does not 
reach the usual high figures. 

In rickets a well-marked lymphocytosis is the rule, which is both 
relative and absolute ; the same holds good for congenital syphilis 
and for the secondary stage of the acquired disease. 

In bronchopneumonia there is at times a well-marked lympho- 
cytosis instead of a polynuclear hyperleucocytosis. Cabot cites an 
instance with a total leucocyte count of 94,600 and 66 per cent, of 
lymphocytes. 

In measles there is at first an increase of the polynuclear neutro- 
philic elements ; later the lymphocytes increase in inverse proportion 
to the neutrophiles, the total number being largely dependent upon 
the degree of glandular involvement. 

In typhoid fever a relative lymphocytosis begins about the end of 
the first week and reaches its highest point in the stage of defer- 
vescence (see page 96). Ewing states that he has found a uniform 
relation in this disease between the lymphocytosis in the blood and 
the grade of lymphatic hyperplasia found at autopsy. He records 
an instance in which the examination of the blood led to a strong 
suspicion of lymphatic leukaemia, and in which at autopsy the mesen- 
teric glands were of unusually large size, and the edges of the partly 
necrotic intestinal ulcers rose 1.5 cm. above the mucosa. 

In uncomplicated cases of pseudoleukemia an absolute increase of 
the leucocytes does not occur ; but there is usually a relative increase 
of the lymphocytes of such extent that the normal ratio to the 

x De Amicis and Pacchioni, Clin. Med. Ital., 1899, No. 1. 
2 Wanstall, Am. Med., 1902. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 107 

polynuelears 1 : 3 rises to 2-3 : 1. This relative lymphocytosis 
Ehrlich and Pinkus regard as characteristic of true pseudoleukemia 
in the differential diagnosis from sarcomatosis and other lympho- 
matous growths. 1 Grawitz, 2 on the other hand, maintains that from 
the leucocyte count no diagnostic conclusions can be drawn, and cites 
cases in which the ratio was either normal or in which the lympho- 
cytes were actually diminished. 

The highest grade of lymphocytosis is met with in the so-called 
lymphatic formr of leukcemia. As in the myelogenous variety, the 
total number of the leucocytes is here also very much increased, 
though not to the same extent. The highest count in Cabot's series 
was 220,000 and the lowest 40,000, so that we may regard 130,000 
as an average. The lymphocytes usually number more than 90 per 
cent. In the chronic cases the small lymphocyte prevails, while in 
the acute cases the large lymphocyte controls the blood picture. 
When septic complications develop, the total number of the leuco- 
cytes, as in the myelogenous form of leukaemia, likewise undergoes a 
considerable diminution, but the lymphocytes still remain relatively 
increased. In one case of Cabot's, in which as the result of septi- 
caemia the total number of leucocytes fell to 471 per cbmm,, the 
percentage of lymphocytes still was 94.7. 

An experimental lymphocytosis has been observed following the 
injection of tuberculin and of extract of carcinomatous growths 
(Grawitz). Waldstein claims to have produced a marked increase 
of the lymphocytes by hypodermic injections of pilocarpin, but, 
according to Ewing, this increase is only relative and brought about 
by a diminution of the polynuclear cells. Wilkinson speaks of a 
lymphocytosis following injections of quinine hydrochlorate, and 
Perry has noted the same after the administration of thyroid extract. 3 

Lymphopenia. — Lymphopenia is notably observed in the acute 
infections which are associated with an increase of the polynuclear 
neutrophiles, and is almost always relative. The condition per se 
has received but little attention, and is as yet unimportant from the 
clinical standpoint. 

Clinical Variations in the Number of the Large Mononuclear 
Leucocytes. — Variations in the number of the large mononuclear leu- 
cocytes are as a rule not sufficiently marked to cause either a distinct 
increase or decrease of the total number of the leucocytes. One notable 
exception to this rule, however, exists in the cases of the acute type 
of lymphatic leukaemia, in which the predominant cell is the large 
lymphocyte, viz., the juvenile form of the common large mononuclear 

1 Pinkus, Die Leuksemie, Nothnagel's Encykl. 

2 Grawitz, Klinische pathol. d. Blutes, 2d ed. 

3 Cited by Da Costa. 



108 THE BLOOD. 

leucocyte, in the sense of Pappenheim. At the same time it must 
be noted that some cases of chronic lymphatic leukaemia also occur 
in which the large mononuclear leucocyte and Ehrlich's transition- 
form represent a large percentage of the leucocytes. These relations, 
however, are not constant. 

In the so-called pseudoleukemia infantum of v. Jaksch a 
marked increase of the mononuclear elements is observed in a 
certain percentage of cases, but in the larger number the general 
increase of the leucocytes is referable to an increase of the poly- 
nuclear cells. 

A relative as well as an absolute increase of moderate grade is 
observed in many of the diseases in which the lymphocytes are 
increased, as in rickets, syphilis, measles, scarlatina, smallpox, etc. 
It is often marked in chronic malaria, and is sometimes seen after 
removal of the spleen. I have observed a marked increase in 
a case of Addison's disease a few days before death, and found 
notable numbers in debilitated individuals, in association with slough- 
ing epithelioma, etc. 

Clinical Variations in the Number of the Mast- cells. — A small 
number of mast-cells is found in the blood under normal conditions. 
The presence of more than 1.5 per cent, is probably always patho- 
logical. A remarkable increase is noted in the myelogenous type 
of leukaemia, and is one of the most constant features of the disease ; 
more constant, in fact, than the increase of the eosinophiles. In 
the one case which I have reported in which the latter were practi- 
cally absent the absolute increase of the mast-cells was well marked 
at the height of the disease. The percentage is not necessarily 
above normal, but not infrequently values of from 5 to 10 per cent, 
are found. It is noteworthy that this increase of the mast-cells 
may be demonstrable at a time when the disease is apparently quies- 
cent ; in one instance of this kind the total number of the leucocytes 
had been 350,000 ; three months later I counted but 2080, of which 
10.9 per cent, were mast-cells. 

A more moderate increase is noted in many other diseases. Gen- 
erally speaking, my experience has been that they are more numerous 
in conditions in which the eosinophiles are also increased, and are 
generally diminished when the eosinophiles are below normal. This 
rule, however, is not absolute. I have found values above the 
normal in various skin diseases, in gonorrhoea, in certain cases of 
malignant disease, associated with eosinophilia. In one case of renal 
carcinoma a few weeks after the removal of the growth I counted 
more than 2 per cent, of mast-cells, with but 1.9 per cent, of 
eosinophiles. Immediately before operation the count had been only 
0.6 per cent. In a case of carcinoma of the cervix I found 0.9 per 
cent, of mast-cells, with 10 per cent, of eosinophiles ; in an advanced 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 109 

case of phthisis I found 1.2 per cent. Canon 1 reports an increase 
of mast-cells in chlorosis ; Sherrington, 2 in cases of Asiatic cholera, 
dying in the reactive stage ; Taylor, 3 in 2 cases of septic bone dis- 
ease ; and Da Costa states that an increase has also been observed 
in some cases of splenic anaemia. 

I have found the number diminished or entire absence of mast- 
cells in some cases of malignant endocarditis, appendicitis, empyema, 
influenza, tonsillitis, intestinal obstruction, lumbar abscess, periproc- 
titic abscess, pernicious anaemia, haematoma of the abdominal walls, 
diabetes, carcinoma of the cervix (septic), " black " jaundice, pneu- 
monia (unresolved). 

Myelocytosis. — At birth and in young children it is usual to 
meet with a small percentage of neutrophilic myelocytes in the circu- 
lating blood under perfectly normal conditions. In adult life, 
however, their presence is always a pathological event. In small 
numbers they may then be met with under the most diverse condi- 
tions. Turk has shown that they are quite common in the acute 
infectious diseases of childhood, and in diphtheria Engel 4 ascertained 
that they are especially numerous in the severe cases (3.6—16.4 per 
cent.). In mild infections they are not usually seen, and when 
present they are found in only very small numbers. In pneumonia 
they are absent or very few in number at the beginning of the dis- 
ease, while at the time of the crisis or immediately thereafter they 
become more numerous and in some cases represent 12 per cent, of 
all neutrophilic cells ; such high percentages, however, are rather 
uncommon and are more apt to be encountered in children than in 
adults. In acute septic conditions a small number of myelocytes 
may also be observed ; larger numbers are found in the more chronic 
cases, which are associated with marked anaemia. In a case of 
lumbar abscess which had been discharging for six months I found 
7.8 per cent. 

In anaemic conditions of whatever origin it is common to meet 
with a moderate number of neutrophilic myelocytes. In pernicious 
anaemia they are quite constant in the active stage of the disease ; 
as a rule the values do not exceed 0.5—1 per cent., but at times they 
may reach 7 per cent. In the secondary anaemia associated with 
syphilis and malignant disease, as also in the pseudoleukaemia of 
v. Jaksch, similar figures are found. In a young child in which a 
notable anaemia had developed as the result of amoebic dysentery 
Amberg counted 9 per cent. In the aestivo-autumnal type of 
malaria they are quite common. 

I have found 2.2 per cent, of neutrophilic myelocytes in a case of 

1 Canon, Deutsch. med. Woch., 1892, vol. xviii. p. 206. 

2 Sherrington, Proc. Royal Soc. London, 1894, vol. lv. p. 189. 

3 Taylor, Contribution from the William Pepper Laboratory, Phila., 1900, p. 148. 
* Engel, Deutsch. med. Woch., 1897, vol. xxiii. No. 8. 



110 THE BLOOD. 

u black " jaundice. Neasser has noted their presence in asphyxia 
and acute mania; Ewing states that they have been found in 
considerable numbers in rickets, osteomyelitis, and osteomalacia. 
Da Costa speaks of their occurrence in poisoning by carbon monoxide, 
in hepatic cirrhosis, acute gout, malignant endocarditis, and exoph- 
thalmic goitre. 

The neutrophilic myelocytes which are met with under these 
various conditions are almost without exception of the small trachy- 
chromatic variety. The amblychromatic variety is practically only 
encountered in the myelogenous type of leukaemia, which is really 
the disease in which large numbers of myelocytes of all kinds find 
their way into the blood. Upon their presence in numbers exceeding 
those found in all other diseases the diagnosis is largely dependent. 
The blood state is that of a true myelcemia. The number of neutro- 
philic myelocytes in myelogenous leukaemia is often most remarkable, 
and a count of from 50,000 to 100,000 per cbmm. is by no means 
exceptional. The average percentage of 18 cases reported by Cabot 
was 37.7, corresponding to a total number of 162,000 leucocytes. 
Coincidently with the neutrophilic myelocytes eosinophilic myelocytes 
also appear in the blood and constitute the majority of the eosino- 
philic cells seen in this type of the disease ; their percentage, 
however, is rarely large. The total number of the polynuclear 
eosinophils is at the same time increased, although the relative 
percentages may be normal or even slightly below normal. The 
polynuclear neutrophilic cells and the lymphocytes, while absolutely 
increased, are relatively much diminished. Of the latter, only 7.6 
per cent, are found on an average, and of the former 49.2 per cent., 
as compared with 20-30 and 60-70 per cent., respectively, under 
normal conditions. The mast-cells, as I have pointed out, are inva- 
riably present in increased numbers in the myelogenous type of the 
disease. 

While the majority of the neutrophilic and eosinophilic cells 
present a normal habitus, it is common in myelogenous leukaemia to 
meet with dwarfed forms of doubtful origin. Occasionally leucocytes 
are observed which are undergoing mitosis. Of special interest is 
the fact that in certain chronic cases of the disease the neutrophilic 
cells apparently lose the power of forming neutrophilic material. 
Non-granular polynuclear cells and myelocytes then appear in the 
blood and may give rise to much confusion in a differential count. 
In one case of this kind reported by Ehrlich the great majority of 
the mononuclear elements, which constituted 70 per cent, of the total 
number, were entirely free from neutrophilic granules. 

The total number of the leucocytes in myelogenous leukaemia in 
the active stage of the disease is much increased. In Cabot's 
series of 30 cases the average was 438,000. If at the same time, 
as not infrequently occurs, there is a coincident anaemia with marked 



MICROSCOPICAL EXAMINATION OF THE BLOOD. Ill 

diminution of the red cells the ratio between the whites and reds 
may fall to 1 : 2 or even 1:1; there are cases on record, indeed, in 
which the leucocytes outnumbered the red cells. Formerly much 
stress was laid upon this ratio in the diagnosis of the disease ; leu- 
kaemia was regarded as a hyperleucocytosis in which the ratio 
exceeded a definite proportion that was generally placed at 1 : 50. 
As a matter of fact, there is probably no other disease in which so 
great an increase of the leucocytes is observed, and even at the 
present day the diagnosis is usually justifiable when an increase of 
such proportions is noted. But, as I have pointed out, myelogenous 
leukaemia is essentially a rnyelaemia, and not a hyperleucocytosis. 
There are cases, moreover, exceptional to be sure, in which the 
increase of the leucocyte is not so extreme, and I have myself ob- 
served one case in which the total number was only 2080 and the 
ratio of the whites to the reds 1 : 1015. The diagnosis of the dis- 
ease should hence be based primarily upon qualitative changes in 
the morpholgy of the blood and only secondarily upon an increase 
of the leucocytes as a whole. 

When septic complications supervene in the course of the disease, 
the blood condition may undergo marked changes. Thus, in propor- 
tion to the degree of infection the myelaemic picture gradually dis- 
appears and is replaced by that seen in simple septic conditions. 
The polynuclear neutrophiles may then increase to 90 per cent., 
and even more, while the eosinophiles diminish and may almost 
disappear. 

In the purely lymphatic form of leukaemia neutrophilic myelocytes 
are scanty ; there are cases of mixed leukaemia, however, in which 
at some stage of the disease the blood picture is essentially of the 
lymphatic type, while at another period there is a marked myelo- 
cytosis. 1 

Eosinophilic myelocytes, aside from their occurrence in myelogenous 
leukaemia, are comparatively rare. They have been found in the 
pseudoleukemia of infants ; Mendel 2 speaks of their occurrence in 
a case of myxoedema ; Turk 3 reports that they are occasionally 
seen in some of the infectious diseases, and Bignami claims to have 
seen them in pernicious malaria. In one case of post-hemorrhagic 
anaemia referable to a ruptured tubal pregnancy I found 1 per cent, 
of eosinophilic myelocytes, and in a case of myelogenous leukaemia 
in which the eosinophiles were absolutely much diminished, the only 
eosinophile that I could find in many slides was a myelocyte. 

1 For a detailed consideration of the blood-changes in leukaemia see especially: 
Pinkus, " Die Leukaeniie," ISTothnagel's Encycl. Ewing, Clinical Pathology of the 
Blood, Lea Bros. Cabot, Clinical Exam, of the Blood, Wm. Wood & Co. Pappenheim, 
Zeit. f. klin. Med. Hsematologisch Streitfragen, 1903. 

2 Mendel, Berl. klin. Woch., 1896, No. 45. 

3 Turk, Klin. Untersuch. d. Blutes, etc., Wien u. Leipzig, 1898. 



112 THE BLOOD. 



The Plaques. 



In addition to the leucocytes and red corpuscles large numbers of 
small roundish elements are encountered in the blood which measure 
about 3 [a in diameter and are free from coloring-matter (Plate II., 
Fig. 1). They are frequently seen collected into groups resembling 
bunches of grapes. These are the blood-plates or plaques of Bizzozero. 
According to Hayem, they represent red corpuscles in an early stage 
of development, and are themselves derived from leucocytes within the 
lymph-channels. He terms them hcematoblasts. This view regarding 
the origin and fate of the plaques is scarcely shared by any modern 
hsematologist. Lilienfeld, Hauser, Howell, and others regard the 
plaques as disintegration-products of leucocytes, and notably the 
nuclear portion, while still others look upon them as precipitated 
globulins derived in part from the morphological elements of the 
blood and in part originating directly in the plasma. More gen- 
erally accepted is the view expressed by Engel, Bremer, Maximow, 
Pappenheim, and others, according to which the plaques are derived 
from the red cells by extrusion. They are originally contained in 
the interior of the cells as so-called nucleoids, and represent the 
remains of the original nucleus, which has lost its individuality as 
the result of chromatolysis. As a matter of fact, it is possible by 
suitable staining to demonstrate the plaques not only within the 
red cells, but also their extrusion from the cells, so that the erythro- 
globular origin of some of these formations at least can scarcely be 
doubted. Jost, moreover, has shown that in the blood of sheep and 
calf embryos they appear at a time when leucocytes are not as yet 
demonstrable. But, on the other hand, there is a possibility that 
what we generally designate as plaques really does not represent a 
unity, and that some of the elements which resemble the true blood- 
platelets may be of different origin. To a certain extent such ill- 
defined little bodies are without doubt derived from leucocytes by a 
process of plasmorhexis — i. e., by the liberation of small bits of 
protoplasm. This may be observed under the microscope directly. 

Deetjen has recently shown that the true plaques are capable of 
executing amoeboid movements when the blood is placed on a slide 
which has been covered with a thin film of agar containing a cer- 
tain amount of sodium chloride, sodium metaphosphate, and dipotas- 
sium phosphate. He also believes to have demonstrated a nucleus 
in the individual plaque, and concludes that the bodies in question 
do not represent artefacts or products of degeneration, but are in 
reality true cellular elements. 

The agar medium which Deetjen employed is prepared as follows : 
5 grammes of agar are dissolved in 500 c.c. of distilled water by 
boiling. The hot solution is passed through a filter, when every 
100 c.c. of the filtrate are treated with 0.6 gramme of sodium 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 113 

chloride, 6 to 8 c.c, of a 10 per cent, solution of sodium meta- 
phosphate, and 5 c.c. of a 10 per cent, solution of dipotassium 
phosphate. To demonstrate the nucleus in smears, the specimens 
are fixed from one to two minutes in 96 per cent, alcohol ; they are 
allowed to dry, and are further fixed from three to five minutes in an 
0.5 per cent, solution of formalin. They are then washed in w T ater 
and stained with hsematoxylin according to Ehrlich's method. I 
have personally found that in ordinary smears which have been 
stained with the eosinate of methylene-blue without any special 
previous fixation the nucleus-like body can also be seen. Quite 
significant is the fact that with any one modification of the Roman- 
owsky method a distinct chromatin stain can be produced in the 
plaques. 

According to Osier, the number of plaques varies normally 
between 200,000 and 500,000 per cbmm. Brodie and Russell, on 
the other hand, claim that this number is too small, and that with 
their improved method of counting (page 147) an average of 635,300 
is obtained. The normal ratio between the plaques and the red cor- 
puscles would thus be 1 : 7.8, taking 5,000,000 as the average nor- 
mal for the red cells. 

Under pathological conditions the plaques may be increased or 
diminished. Hayem's statement that they occur in greatly dimin- 
ished numbers in pernicious anaemia has on the whole been con- 
firmed. According to Lazarus, van Embden found 64,000 and 
32,000 in two cases, which, accepting the normal figures of Russell 
and Brodie, would mean a diminution to less than one-twentieth of 
the normal. At times they may indeed be absent, but in some cases 
increased numbers are also found (Strauss and Rohnstein). In leu- 
kaemia the plaques are often greatly increased. A large increase is 
at times observed in post-hemorrhagic anaemia and in chlorosis, but 
the results are not constant. In the secondary anaemias referable 
to carcinoma, sepsis, tuberculosis, etc., the findings are variable ; 
sometimes an increase is observed, at others a decrease, and then 
again normal values ; the results, moreover, are inconstant in one 
and the same case. In the acute infectious diseases their number 
is the smaller the more severe the course of the disease. 

To study the plaques in the wet preparation it is necessary to 
prevent their agglutination by the immediate addition of a drop of 
Hayem's fluid (page 141), or the reagent suggested by Afanassiew, 
which has the following composition : 

Peptonum siccum . . . 0.60 

Methyl-violet ...» . . 0.01 

Physiological salt solution (0.9-0.95 percent.) . .100.00 

The reagent must be kept sterile. Either method will answer 
most purposes, and it is well to puncture the finger through a drop 
8 



114 THE BLOOD. 

of the fluid. If it is desired to study the movements of the plaques, 
Deetjen's method must be employed. In the dry preparation they 
are most conveniently demonstrated with the eosinate of methylene- 
blue or one of the modifications of the Romanowsky method. 
(For the enumeration of the plaques see page 147.) 

Literature.— Bizzozero, Virchow's Arcliiv, vol. xc. Hayem, Le sang, Paris, 
1889. Howell, Jour, of Morph., 1891, vol. iv. p. 57. Maxiniow" Arch. f. Anat., 1899, 
vol. i. p. 33. Jost, Arch. f. mik. Anat., 1903, vol. lxi. p. 667. Determann, Deutsch. 
Arch. f. klin. Med., vol. lxi. p. 365. Deetjen, Virchow's Archiv, 1901, vol. clxiv. p. 
239. Brodie and Russell, Jour. Physiol., 1897, Nos. 4 and 5. 

The Dust Particles or Haemokonia of Miiller. 

These may be seen in any fresh specimen of blood mounted in the 
usual manner. They are small, generally round, sometimes dumb- 
bell-shaped, colorless, highly refractive granules, which manifest 
very active molecular movements. They occur in the plasma of the 
blood and are apparently not connected with the process of coagula- 
tion. Miiller found them abnormally numerous in a case of Addi- 
son's disease, while they were diminished during starvation and in 
various cachectic conditions. Stokes and Wegefarth regard these 
granules as identical with the neutrophilic and eosinophilic granules 
of the leucocytes. They suppose that the bactericidal power of the 
leucocytes and of the serum of man and many animals is due to their 
presence. As a matter of fact, the origin of the hremokonia from 
the granular leucocytes can not infrequently be directly observed. 

I have quite constantly found the haemokonia increased at the 
height of digestion, and have then repeatedly observed their extru- 
sion from both neutrophilic and eosinophilic cells. 

Literature. — H. F. Miiller, " Ueber einen hisher nicht beach teten Formbestand- 
theil d. Blutes," Centralbl. f. allg. Path. u. path. Anat., 1896, p. 929. W. E. Stokes 
and A. Wegefarth, "The Presence in the Blood of Free Granules, etc., and their 
Possible Eelation to Immunity," Johns Hopkins Hosp. Bull., 3897, p. 246. E. B. 
Sangree, " Leucocytic Granules," etc., Phila, Med. Jour., 1898, p. 472. 

General Technique. 

Slides and Cover-glasses.— To obtain satisfactory results, it is 
essential to have glassware of the best quality. The cover-glasses 
should not measure more than 0.08-0.10 mm. in thickness and must 
be cleansed with care. The same holds good for the slides, which 
should have a level surface ; many of those furnished by dealers 
are unsatisfactory for work with immersion lenses. 

Both covers and slides should be placed in concentrated sulphuric 
acid or in glacial acetic acid for several hours. They are thoroughly 
washed in running water and distilled water and then placed in 
alcohol and finally in ether, where they remain for several hours. 
During this process care must be had that they are well separated 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 115 

from each other. Subsequently they are kept in jars with absolute 
alcohol, and are dried just before use, or they may be dried at once 
with fine linen or Japanese lens paper and stored in dust-proof 
receptacles. When once cleansed, the cover-glasses should be 
handled only with forceps, as the moisture of the hands is in itself 
sufficient to produce post-mortem changes in the red corpuscles. 

To cleanse slides that have been used, the covers must first be 
removed by immersion for several days in xylol or turpentine. 
They are then placed in hydrochloric acid to which about a tea- 
spoonful of potassium chlorate has been added for every 30 c.c. 
The mixture is kept on the boiling water-bath to the point of 
decolorization. The slides are next rinsed in hot water, heated for 
a half hour in a thin mush of equal parts of washing soda, sawdust, 
and talcum, prepared with the aid of water and stirring frequently, 
then washed off with hot water acidified with hydrochloric acid, and 
finally with pure hot water, alcohol, and ether. 

The Blood Mount. — We distinguish between wet mounts and 
dry mounts. Wet specimens can only be utilized successfully if the 
patient is near at hand to the laboratory, as in office-work and in the 
hospital ; where several hours must elapse before the preparation can 
be examined, it will usually be best to resort to the dry specimen. 
Wet preparations, however, are very convenient and yield a large 
amount of information without delay, and a rapid survey will indi- 
cate whether or not it will be necessary or advisable to resort to a 
more detailed examination. The grade of an anaemia, the degree, 
character, and extent of a hyperleucocytosis, the presence of malarial 
organisms, can all be told from the wet preparation. With the dry 
and stained specimen, on the other hand, all these points are brought 
out more distinctly, and other information is further afforded which 
cannot be obtained from the wet specimen alone. 

To prepare a blood specimen, the tip of a finger, or in children 
especially the lobe of the ear, is first cleansed with ether and then 
punctured with a suitable instrument, such as a fine lancet or a 
stout needle. The puncture should be sufficiently deep that the 
blood will flow from the wound without undue pressure. 

To prepare a wet specimen^ a clean cover-glass is taken up with a 
pair of forceps, with flat blades and a light spring, touched to the 
drop without coming in contact with the skin, and immediately 
transferred to a clean slide. If suitable glassware is used that 
is perfectly clean, the drop will immediately spread in a circular 
fashion between cover-glass and slide, and on examining with a low 
power, which should always precede examination with a high power, 
it will be noted that in the central portion of the specimen especially 
the red cells will be well separated from one another and will not 
have run into rouleaux. This will only occur if the glassware is 
imperfect, if it is not perfectly clean, or if the drop has been too 



116 



THE BLOOD. 



large. To gauge the proper size of the drop requires a little practice. 
Along the margin of the specimen, where a certain amount of evap- 
oration is going on, it is usual to find rouleaux and crenated red 
corpuscles, even though the rest of the specimen is perfect, and in 
the course of time post-mortem changes will also become noticeable 
throughout the preparation. If the specimen is ringed with a little 
paraffin, however, a satisfactory examination is still possible after a 
number of hours, and even without being ringed such preparations 
can be kept for at least one hour. 

To prepare dry specimens, which are subsequently to be stained, 
the blood is spread in a capillary layer between cover-glasses or on 
slides. In the first case, one cover-glass is locked in a pair of 
forceps such as those devised by Ehrlich and pictured in the accom- 
panying illustration (Fig. 15). A second cover is taken up with 

Fig. 15. 




Ehrlich' s cover-glass forceps. 

a pair of forceps without a lock, but with flat blades and a light 
spring ; this is held to the drop of blood just as it emerges from the 
puncture, and is then immediately laid upon the first cover. If the 
glasses are of satisfactory quality and clean, the blood will at once 
spread in a capillary layer ; the top cover is then drawn from the 
lower cover by grasping the edge firmly with the fingers and making 
even traction in a plane parallel to the other. Here also a certain 
amount of experience is necessary in ganging the size of the drop 
in reference to the size of the covers. In no case should it be so 
large that the top cover floats upon the blood. If the drop is rather 
small, the two covers should overlap only to such an extent as to 
furnish a space which is just filled by the blood. If the drop is 
larger, they should overlap over a larger surface. 

The above method is the one originally suggested by Ehrlich, and 
probably the one most commonly employed for making dry smears. 
Personally I have almost abandoned the use of cover-glasses, 
and much prefer slides for routine work. But little practice is 
required to obtain very satisfactory results, and it is possible to 
control the quality of the individual smears with a degree of pre- 
cision which is but rarely attained even by the most experienced 
workers with cover-glasses. The spreads, moreover, are much 
larger, so that there will always be a sufficient number of leucocytes 
available even under normal conditions to permit a count of at least 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 



117 



a thousand cells. At the same time it is possible to spread portions 
of the drop so thin that the individual cells are well separated the 
one from the other, while other portions can be made a little thicker. 
The slides are cleansed in the same thorough manner as in the case 
of the cover-glasses. A fair-sized drop of blood is then mounted 
near the end of one slide and spread with an even sweep with the 
edge of a second slide; this should be done with a light hand, 
and holding the first slide in the left hand between the thumb and 
the second and third fingers. The second slide should also be held 
in this manner, but at an angle of 45 degrees to the first, as shown 
in the accompanying illustration (Fig. 16). Before commencing the 

Fig. 16, 




The preparation of blood-smears on slides. 

sweeping movement I let the blood spread along the edge of the 
second slide by capillary attraction; then I move across, gradually 
raising the second slide to a vertical position. Pressure must be 
carefully avoided. 

After being allowed to dry in the air the specimens are placed 
between layers of filter-paper and may then be examined at leisure. 
If several days must elapse before the examination, it is well to 
place them, wrapped in filter- paper, in closed jars. Should it be 
desired to preserve the specimens for a long time — i. e., for months 
or years — it is best to coat the films with a thin layer of paraffin, 
which later is dissolved by immersion in toluol. In this manner 
especially valuable and rare specimens may be kept almost indefi- 
nitely. Unless this precaution is taken, the staining qualities of all 
the morphological elements of the blood will undergo changes which 
render the specimens unfit for color analysis. 



118 TH.E BLOOD. 

Fixation. — Before staining, it is frequently necessary to fix the 
blood-films, to which end several methods may be employed. The 
best results are usually obtained by heat. For this purpose a 
copper plate may be used measuring about 10 cm. in width by 40 
cm. in length and 3-5 mm. in thickness ; this is heated by a Bun- 
sen burner or a small coal-oil stove. After the plate has a fairly 
constant temperature, the desired degree is ascertained by a series 
of drops of water, toluol (boiling-point, 110°-112° C), or xylol 
(137°-140° C), etc., noting the line at which ebullition occurs. If 
the distance of the plate from the flame and the size of the flame, 
etc., are constant, the apparatus requires practically no attention and 
serves its purpose very well. As a rule a brief fixation only is 
necessary — i. e., exposure to a temperature of from 100° to 126° 
C, for one-half to two minutes, while in special cases Ehrlich recom- 
mends a more prolonged exposure or a higher temperature. Very 
good results are obtained for most purposes by heating the blood- 
films to a temperature of 140° C. for thirty to forty-five seconds, 
as suggested by Rubinstein. This point is conveniently ascertained 
on the copper plate by noting the line at which the so-called Leiden- 
frost phenomenon begins to occur, viz., the point at which a drop of 
water assumes the spherical form and rolls about on the plate. 

In the place of the copper plate an ordinary drying-oven provided 
with a thermostat and thermometer or a so-called Victor Mayer 
Siedekessel may also be employed. The latter is a small copper 
kettle covered with a thin plate, which is perforated for the recep- 
tion of the boiling-tube. If a small quantity of toluol is boiled 
in this kettle for a few minutes, the copper plate will acquire a 
temperature of from 107° to 110° C, and retains this sufficiently 
long for ordinary purposes (Ehrlich). 

Absolute alcohol or a mixture of equal parts of absolute alcohol 
and ether (Nikiforoif ) have also been recommended as fixing agents 
for blood-films, but are not very satisfactory for the study of the 
neutrophilic granulation. With Ehrlich's triacid stain especially it 
will frequently be noted that the granules are stained imperfectly or 
not at all. For the study of nuclear structures, however, both are 
quite satisfactory. In the case of absolute alcohol alone immersion 
of the blood-films for a few minutes is sufficient ; with alcohol and 
ether fixation for one-half to two hours is necessary. 

Formalin is useful as a fixing agent and may be used in con- 
nection with practically all the common blood-stains. A 1 per 
cent, alcoholic solution is employed. This is prepared by diluting 
one part of the commercial formalin, which is a 40 per cent, 
solution of formaldehyde in gas, water and methyl alcohol, with 
nine times its volume of water, and one part of the resulting solution 
with nine times its volume of alcohol. Fixation is completed in 
one minute, and for practical purposes it is merely necessary to cover 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 119 

the blood-films with a few drops of the solution, which is then 
drained off and replaced with the staining reagent directly. 

With certain staining reagents, such as Jenner's eosinate of 
ruethylene-blue, or with Leishman's method, previous fixation is not 
necessary, as the films are here fixed by the methyl alcohol during 
the process' of staining. 

The Anilin Dyes and the Principles of Staining. — The anilin 
dyes with which we have to deal in the clinical laboratory are all 
derivatives of hydrocarbons and for the most part of hydrocarbons 
of the aromatic series. Their staining properties are dependent 
upon the presence in the individual compounds of two distinct 
atomic complexes which are spoken of as chromophobe and auxo- 
chromic groups, respectively. The presence of the chromophoric 
group imparts chromogenic properties to the substance, the dye itself 
resulting on the further introduction of an auxochromic group. The 
auxochromic groups are salt-forming radicles and render the dye 
either basic or acid. Two markedly auxochromic radicles are known, 
viz., the strongly basic amido group — NH 2 and the feebly acid 
hydroxy 1 group — OH. Still other salt-forming radicles may enter 
into the composition of the dye, but it is noteworthy that these have 
but feebly developed auxochromic properties. Radicles of this order 
are notably the carboxyl group — COOH, the sulphoxyl group 
— S0 2 OH, the nitro group — N0 2 , and the nitroso group — NO 
(which two latter may also occur as chromophoric radicles). These 
groups are essentially of influence upon the reaction of the dye, and 
as the chromophoric radicle itself may have acid or basic tendencies 
it is manifest that the ultimate reaction of the individual compound 
will depend upon the inter-relation of acid and basic radicles. 
Markedly acid dyes will result if both the chromophoric group and 
the salt-forming radicles are acid, while strongly basic dyes will be 
the outcome if both have basic tendencies. Between these two 
extremes various possibilities exist, the ultimate reaction depending 
upon the character of the chromophore, the presence of acid or basic 
salt-forming radicles, the simultaneous presence of both, their num- 
ber, etc. We may accordingly divide the various dyes into the fol- 
lowing classes : 
. 1. Basic amido dyes. 

2. Acid nitroso dyes. 

3. Acid sulpho- and nitro-dyes, viz., amido- or oxysulphonic 
acids, amiclo-oxysulphonic acids, nitrophenols, nitroamins, nitro- 
amidosulpho acids, nitro-oxysulpho acids, nitroamido-oxysulpho acids. 

4. Acid oxy- and oxycarbonic dyes. 

5. Amido-oxy-, amidocarbonic, and amido-oxycarbonic dyes. 

6. Amidosulphocarbonic-, oxysulphocarbonic-, amido-oxysulpho- 
carbonic-, amidonitrocarbonic-, oxynitrocarbonic-, amido-oxynitro- 
carbonic-, and amido-oxysulphonitrocarbonic dyes. 



120 THE BLOOD. 

Of chroinophoric groups, some twenty are known, and it is cus- 
tomary to classify the anilin dyes on the basis of these underlying 
radicles. We thus find : 

The — N0 2 group in the nitro dyes (picric acid, Martius yellow, 
naphtol-yellow S, aurantia). 

The — NO group in the nitroso dyes (Echtgrun naphtol-green). 

— N=N — in the azo dyes (anilin-yellow, chrysoidin, vesuvin, 
Sudan G and III, alizarin-yellow FS, Ponceau, Bordeaux, amaranth, 
coccinin, orange G, tropseolin, Biebrich scarlet, congo, benzopur- 
purin) : 

/ in the rosanilins (malachite-green, brilliant green, methyl- 

^\" II violet, methyl-green, fnchsin, acid fuchsin, iodine- 

green, anilin-blue, alkali-blue, water-blue, aldehyde- 
green). 

C\~ in the rosolic acid dyes (aurins). 

C\~ in the phthaleins (eosin, spriteosin, erythrosin, phloxin, 

rose bengale, rhodamin, gallein, ooerulein). 

^CXX in the anthraquinons (alizarin, purpurin, anthragallol, alizarin- 
\ co / blue). 



N\ in the indamins (phenylene-blue, Bindschedler's green, 

J 



toluylene-blue). 



I ^ R— o in the indophenols (indophenol-blue). 

N\ ^S in the Lauth dyes (Lauth's violet or thionin, methylene- 
j blue, methylene-red, methylene-green). 

N= 

— N— in the azins (eurhodin, eurhodol, toluylene-red, the safranins, 
_N_ Magdala red, mauvein). 

X \po * D euxan thinic acid and possibly in galloflavin (jaune 
\r/ indienne). 

in the quinolins and acridins (cyanin, quinolin-red, quinolin- 
V/ yellow, acridin-red and scarlet). 

The majority of the anilin dyes are found in the market in the 
form of salts of the respective staining acids and bases, and it is 
noteworthy that the latter as such are for the most part either 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 121 

colorless or but feebly stained. Triamidotriphenylcarbinol is thus ' 
colorless, while its monacid salts are red (fuchsin) ; phenolphthalein 
likewise is colorless, but forms red salts with the alkalies ; fluorescein 
is pale yellow, but forms the bright-red fluorescent uranin with 
alkali, etc. The phenols and nitrophenols, however, are commonly 
used as free acids. 

During the process of staining, the salts of the staining acids or 
bases are decomposed by the animal or vegetable tissue and new 
compounds result between the free staining acid or base and the 
various chemical components of the tissue in accordance with the 
reaction of its component parts. The acid nuclear substance of cells 
thus shows a special affinity for basic dyes, and the basic proto- 
plasm for acid dyes. Contrasted with this chemical process of 
staining is the physical process in which the dye is merely stored in 
the pores of the tissue. Both must be sharply differentiated the one 
from the other in attempting to draw inferences in reference to 
chemical affinity on the part of component parts of a tissue or a cell. 

While in former years simple dyes were commonly employed in 
the clinical laboratory and tissues were stained successively if more 
than one dye was used, it has been shown that it is possible to com- 
bine acid dyes with basic dyes in such manner that the acid affini- 
ties of the one become completely saturated by the basic affinities of 
the other. Neutral dyes thus result in which the staining possibili- 
ties of the two original components are not only preserved, but in 
which additional staining properties have also developed which are 
the expresssion of their neutral reaction. Stains of this order with 
well-developed polychrome properties are, of course, exceedingly 
valuable, as they readily permit an insight into the structure and to 
a certain extent into the chemical composition of cells which is 
otherwise only obtained with much difficulty. The credit of having 
first prepared and used such neutral dyes belongs to Ehrlich, whose 
triacid stain has been and is still one of the most important differ- 
ential dyes employed in the clinical laboratory. 

The principle underlying the formation of neutral dyes of this 
character is well shown in the case of the triacid stain. Three single 
dyes enter into its composition, of which two are acid dyes, viz., 
acid fuchsin and orange G, while the third, methyl-green, is a basic 
dye. This latter contains three basic groups which may be saturated 
by corresponding acid groups. The term " triacid " has thus not 
reference to the presence of three acid dyes, but to the fact that the 
three basic radicles of the basic component of the dye have been 
saturated in the manner indicated. In the present instance these 
three radicles have not been neutralized by one acid dye, but by two. 
As a result we have present in the same mixture a fuchsinate of 
methyl-green and the corresponding neutral compound of the orange 
G and methyl-green. Solutions of both can be directly mixed, the 



122 THE BLOOD. 

one with the other, which is always possible with solutions of neu- 
tral dyes if the two solutions to be mixed have one component in 
common. 

While the simple dyes, both basic and acid, are soluble in water, 
the neutral dyes are practically insoluble, but soluble in an excess 
of either the acid or the basic component, and more especially the 
former. If then an aqueous solution of methyl-green is added 
carefully to an aqueous solution of acid fuchsin, fuchsinate of 
methyl-green is formed at once, but at first remains in solution 
owing to an excess of the acid dye. Upon the further addition of 
methyl-green, however, and standing, a point is reached when the 
fuchsinate separates out, and if the amounts of the two components 
have been carefully determined beforehand the filtrate may be nearly 
colorless. If then an excess of methyl-green is added, a certain 
amount of the fuchsinate will redissolve ; and if the excess be 
sufficiently great, the entire precipitate will pass into solution. 

Aside from an excess of the acid or basic component of the 
neutral dye its solution can also be brought about in other ways, as 
with alcohol (notably methyl alcohol), acetone, methylal, etc. 

Not all simple dyes are equally well adapted for the preparation 
of neutral dyes. Of basic dyes, the most useful are those which 
contain the so-called ammonium group, notably methyl-green, 
methylene-blue, amethyst-blue, and to a certain extent also pyronin 
and rbodamin ; of acid dyes, the readily soluble salts of the polysul- 
phonic acids, such as orange G, acid fuchsin, and narcein, and of 
the salts of the carbonic acids eosin. 

Neutral mixtures may then be prepared which contain two or 
more component dyes. If it is desired to prepare a tricolor mixture, 
two possibilities suggest themselves, viz., a mixture containing one 
acid dye and two basic dyes, or one with one basic dye and two acid 
dyes. 

The principle of staining with neutral dyes is the same as in the 
case of the simple acid or basic dyes. Taking the leucocytes, for 
example, the nucleins will be found to decompose the neutral body 
and to unite with the basic component ; the eosinophilic granules 
similarly decompose the dye, but take up the acid component, while 
in the case of the neutrophilic granules we may imagine that no 
decomposition is here effected, but that the neutrophilic material 
unites directly with the neutral molecule. 

The number of neutral dyes in use in the clinical laboratory is as 
yet small ; several are modifications of Ehrlich's original triacid. 
In Pappenheinr's two triacid mixtures methylene-blue is used as the 
base in the one, and methylene-azure as the base in the other. 

To the class of the neutral dyes also belongs the eosinate of 
methylene-blue and the eosinate of methylene-azure which form the 
basis of the various eosin-methvlene-blue solutions originally sug- 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 123 

gested by Ronianowsky, Ziemann, Chenzinsky, Plehn, Ehrlich, 
Michaelis, etc. The eosinate of methylene-blue solution is commonly 
spoken of as Jenner's stain ; the eosinate of methylene-azure forms 
the basis of the various modifications of the Romanowsky stain, 
such as those suggested by Nocht, Ziemann, Leishman, Reuter, etc. 
All these stains are of the utmost value in the morphological study 
of the blood more particularly, and although many are merely 
modifications the one of another, I have described their preparation 
and uses in detail, for one will frequently serve to bring out a special 
point which is only well shown in this particular manner. The 
selection of the individual method will, of course, depend upon 
circumstances. For routine work I suggest the eosinate of methylene- 
blue in Jenner's modification, Ehrlich's triacicl, Pappenheim's triacid 
No. 2, Ziemann' s method, and Wright's modification of Leishmau's 
method ; for the study of malarial organisms aside from the routine 
stains (excepting Ehrlich's triacid), notably Nocht's stain, Reuter's 
stain, and Futcher's stain. For the study of the oxyphilic granula- 
tion Ehrlich's triglycerin mixture is essential ; for the investigation 
of mast-cell granules Ehrlich's dahlia, and the various violet basic 
anilin dyes ; for the neutrophilic granulation besides the usual 
routine stains mentioned, also Ehrlich's neutral mixture ; for the 
study of the lymphocytes Pappenheim's pyronin-methyl green, 
while for the study of nuclear structures the old eosin-haematoxylin 
method still is unsurpassed. 

Methods of Staining. 

General Methods. — The Eosinate of Methylene-blue (Jenner). 1 — 
Equal parts of a 1.2—1.25 per cent, aqueous solution of eosin and a 
1 per cent, aqueous solution of methylene-blue are mixed in an open 
basin and allowed to stand for twenty-four hours. The resulting pre- 
cipitate — the eosinate of methylene-blue — is washed with water, col- 
lected on a filter, dried at a moderate temperature, and finely powdered. 
The dye can then be stored in bottles and is perfectly stable. For 
staining purposes an 0.5 per cent, solution in absolute methyl alcohol 
is employed ; this can be used at once and keeps indefinitely. I have 
used this stain as a routine stain for nearly four years and can speak 
definitely of its value. It has material advantages over the triacid stain 
as special fixation is not required and as its power of differentiation is 
more extensive. Its preparation also is simpler, though it is necessary 
to observe certain precautions. In preparing the dye I first weigh out 
the requisite amount of eosin and methylene-blue. The eosin is placed 
in a mortar or evaporating-dish and rubbed into a paste with a small 
amount of water ; more water is then added until all the dye is well 
dissolved. This solution is poured into a large saucepan and diluted 

1 Jenner, Lancet, 1899, vol. i. p. 370. Simon, Maryland Med. Jour., April, 1900. 



124 THE BLOOD. 

to the proper point. The methylene-blue is now similarly brought 
into solution, though with a little more difficulty as the dye is 
inclined to be lumpy ; it must all be dissolved. This solution is 
poured directly into the eosin solution and the requisite amount of 
water further added. The mixture is stirred with a rod and left to 
stand. If the proper quantities have been used and well dissolved, 
the filtrate is but little colored, in which case not much washing is 
necessary ; if, however, there is a distinct excess of either dye, and 
notably the methylene-blue, this must be washed out, which is best 
done by decantation. The alcoholic solution finally is prepared by 
rubbing up the dye with the alcohol in a porcelain dish. Absolute 
methyl alcohol must be used. 

The blood-films (on slides) are not fixed before staining ; this is 
accomplished by the absolute alcohol during the staining. The 
specimens are well covered with the stain and after about five 
minutes washed off with water and dried in the air or by heating 
moderately over a flame. Care should be had during the staining 
that the preparations are thoroughly covered with the dye, as other- 
wise some of the stain is apt to become precipitated as the result 
of evaporation. After drying, the specimens can be examined 
directly in a drop of cedar oil. Permanent mounts are prepared by 
placing a drop of Canada balsam on the specimen and covering it 
with a cover-glass. With the precautions stated and by strictly 
adhering to the method as described even the beginner can obtain 
perfect results. For routine purposes I can recommend the stain 
without reserve. The differentiation is excellent and most extensive 
(see Plates III., IV., and VI. especially). The red corpuscles are 
stained a terra cotta, the nuclei of the leucocytes and nucleated red 
cells blue, the plaques mauve, the neutrophilic granules a purplish 
red, the eosinophilic granules bright red, and the mast-cell granules 
dark violet. Granular degeneration and polychromasia of the red 
cells is well shown (Plate III.). Malarial organisms, bacteria, and 
filaria? are stained blue. 

Ehrlich's Triacid Stain. 1 — The preparation of a reliable triacid 
stain, according to Ehrlich, presupposes the use of chemically 
pure dyes, such as those prepared by the Actiengesellschaft fur 
Anilinfarbstoffe of Berlin. Saturated aqueous solutions of orange G, 
acid fuchsin, and methyl-green are first prepared and allowed to 
clear by standing for at least one week. It is essential that these 
solutions should be perfectly clear, and it is well in measuring off 
the requisite quantities to remove the supernatant portion with a 
pipette. The various ingredients are then mixed in a clean bottle, 
making use of the same measuring-glass, and without washing 
between the addition of the individual components. These are 
taken in succession as shown below, and after adding the methyl- 

1 Ehrlich-Lazarus, Die Anseraie, loc. cit. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 125 

green the mixture is thoroughly stirred until the remaining portion 
of alcohol and glycerin has been added. 

Orange G solution 13.0-14.0 c.c. 

Acid fuchsin solution 6.0- 7.0 c.c. 

Distilled water 15.0 c.c. 

Absolute alcohol 15.0 c.c. 

Methyl-green solution 12.5 c.c. 

Absolute alcohol 10.0 c c. 

Glycerin 10.0 c.c. 

The solution is ready for use at once and does not deteriorate 
with age. 

In order to obtain the best results with the stain, it is practically 
necessary to fix the blood-films by heat ; fixation by Nikiforoff's 
method does not furnish constant results, and only too often leaves 
the neutrophilic granules unstained or imperfectly stained. Fixation 
at a high temperature (140° C), as suggested by Rubinstein fur- 
nishes better results than the lower temperatures originally advised 
by Ehrlich, as the difference in color between the neutrophilic 
granules and the eosinophilic granules is thus brought out more 
prominently. The blood specimens are stained about five minutes, 
then washed in water, dried (by blotting, if desired), and examined 
as usual. 

In properly stained specimens the eosinophilic granules present a 
copper or a yellowish-red color, while the neutrophilic granules are 
violet. The mast-cell granules remain colorless and appear as round 
vacuoles in the faintly bluish-greenish protoplasm. The nuclei of the 
leucocytes present a greenish color and are not well stained. The 
red cells in properly heated specimens are orange ; if the tempera- 
ture was too high, they are yellow, and in such a case it will be 
found that their structure has suffered as a consequence. If the 
temperature has been too low, the red cells take on the fuchsin. 
The nuclei of the normoblasts are intensely stained ; the older nuclei 
appear black ; megaloblastic nuclei, on the other hand, are rather 
feebly stained, and in some specimens, indeed, the inexperienced 
will at first sight not discern any nucleus. Granular degeneration 
is not shown and polychromatophilia cannot be demonstrated so well 
with the triacid as with the eosinate or in ha3matoxylin-eosin prepara- 
tions. Malarial organisms are imperfectly shown. The differentia- 
tion with the triacid is thus markedly less than in the case of the 
eosinate. This is owing to the peculiar character of the methyl- 
green, which is a specific nuclear dye. To counteract some of these 
deficiencies, Ehrlich has suggested to stain the preparations for a few 
seconds with an aqueous solution of methylene-blue first, and to stain 
with the triacid afterward. This improves the pictures somewhat, 
but it is not wholly satisfactory. As a routine stain in the clinical 
laboratory Ehrlich' s triacid is at present less commonly employed ; 



126 THE BLOOD. 

in the special study of the neutrophilic granulation, however, it still 
retains its usefulness. 

Pappenheim's Triacid Stain (No. I.). 1 — This, as well as Pappen- 
heim's triacid No. II., has been devised to utilize the advantages 
of a tricolor mixture while obviating the disadvantages attaching to 
methyl-green as a base. It is prepared as follows : 1 part of 
methylene-blue is dissolved in a small volume of water and treated 
with a concentrated aqueous solution of acid fuchsin, drop by drop, 
until a precipitate forms ; the addition of the fuchsin solution is then 
contiuued while stirring until the precipitate is again dissolved, an 
excess being carefully avoided. The resultant solution is termed A. 
A second solution B is prepared in a similar manner, but starting 
with 4 parts of methylene-blue and using a concentrated aqueous 
solution of orange G as the acid dye. The two solutions are then 
mixed and the specimens stained as with Ehrlich's triacid after being 
fixed by heat. They are washed in water, dried, and examined as 
usual. 

The red cells are orange, all nuclei blue, the neutrophilic granules 
a purplish violet, the eosinophilic granules red, and the mast-cell 
granules a bluish violet. Granular degeneration and polychromato- 
philia are well shown and the malarial organism also is stained. 

Pappenheim's Triacid Stain (No. II.). 2 — This contains the active 
principle of Unna's polychrome methylene-blue as base, which 
Michaelis claims to have identified as methylene-azure, and which he 
terms azure blue. The dye is best purchased, already prepared, 
from Griibler, where it is sold under the name of Pappenheim's 
" panoptic triacid solution." It is used in the same manner as 
Ehrlich's triacid, after previous fixation of the specimens by heat, 
but must always be freshly prepared in aqueous solution. It is 
recommended to give the nuclei a preliminary stain with a concen- 
trated aqueous solution of toluidin-blue, after which the dye is 
washed off and replaced directly by the triacid. 

The advantages of this stain are referable to the peculiar selective 
action of the azure blue for nuclear structures, and the possibility of 
demonstrating the presence of such formations in this manner, where 
with other methods they*cannot be shown. 

The nuclei of the lymphocytes and large mononuclear leucocytes 
and of the malarial organisms are colored a bright red, those of the 
polynuclear leucocytes a bluish violet, and the erythroblastic nuclei 
almost black. The bodies of the lymphocytes and the malarial 
organisms are sky blue, the erythrocytes fuchsin-red or orange 
(according to the degree of heat employed during fixation), the neu- 
trophilic granules violet red, the eosinophilic granules a bright 
scarlet, and the mast-cell granules carmine. In this manner the 

1 Pappenheim, Deutsch. med. Woch., 1901, vol. xxvii. p. 799. 

2 Pappenheim, Ibid. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 127 

presence of chromatin can also be demonstrated in the plaques, 
which for the most part present a red nucleus surrounded by a pale- 
blue protoplasm. 

The Romanowsky Method. 1 — The history of the Romano wsky 
method is intimately associated with the study of the minute structure 
of the malarial organism, in which the presence of a nucleus was 
first demonstrated by its aid. The dye is essentially an eosin- 
methylene-blue mixture, the specific staining action of which is, 
however, not due to the methylene-blue per se, but to a decomposi- 
tion-product of the methyleoe-blue, viz., the methylene-azure. As I 
have stated, this also forms the base of Pappenheim's triacid No. II. 
It apparently combines with eosin to form a neutral dye analogous 
to the eosinate of methylene-blue, and can similarly be employed as 
a routine blood stain in the clinical laboratory. As a rule we do 
not employ solutions of the pure dye, but solutions of methylene- 
blue containing a variable amount of the methylene-azure, to which 
the requisite amount of eosin is at each time added. In a mixture 
of this kind the neutrophilic material is then probably stained in 
statu nascendi. 

Reuter has attempted to prepare a stable solution of the eosinate 
of methylene-azure (with eosinate of methylene-blue) in methyl 
alcohol, with the addition of oil of anil in, analogous to Jenner's 
eosinate of methylene-blue, but unfortunately the solution keeps for 
only a few weeks. After that time so much of the specific dye sepa- 
rates out that not sufficient remains in solution to produce the red 
color in the nuclei of malarial organisms, even though the nuclei of 
the white cells are still stained red. 

Giemsa was the first to devise methods for the economical prepa- 
ration of methylene-azure in pure form, which can be obtained 
from Griibler ; in his method an aqueous solution of the azure is 
directly combined with an aqueous solution of eosin, the two being 
freshly mixed at each time. Here also the neutrophilic material is 
then stained by the eosinate of methylene-azure in statu nascendi. 

The following modifications of the original Romanowsky method 
are based in principle upon the above considerations : 

Nocht's Method. 2 — Two standard solutions are employed, viz., 
a 1 per cent, aqueous solution of eosin which before use is diluted 
with 20 to 50 times its volume of water, and a solution of methylene- 
azure in methylene-blue which is prepared as follows : 100 c.c. of a 
1 per cent, aqueous solution of methylene-blue are treated with the 
precipitated oxide of silver obtained from 1 gramme of silver nitrate 
by dissolving this amount in water, precipitating with caustic alkali 
solution, and washing. After standing for four or five days at 

1 Romanowsky, St. Petersburg, rued. Woch., 1891. 

2 Nocht, Encyk. d. mik. Tech., vol. ii., Urban u. Schwarzenberg, Berlin-Wien, 1903, 
p. 785. 



128 THE BLOOD. 

ordinary temperature the methylene-blue solution has a distinct red- 
dish tone and contains a considerable amount of methylene-azure. 
Such a solution is almost neutral. 

The staining reagents must always be prepared anew by adding 
azure blue solution to the eosin drop by drop while stirring, until 
the color of the resultant mixture is the same as that of the azure 
solution. If the original methylene-blue solution from which the 
azure blue was prepared was of the strength of 1 per cent., not quite 
double the amount of the azure solution as compared with the eosin 
solution will be necessary. As a rule 4 drops of the eosin, 20 c.c. 
of water, and 6 to 8 drops of the azure solution will be found about 
the proper proportions ; an excess of the azure blue does not matter. 

Cover-glass specimens are best used, which should be fixed by heat 
or by immersion in alcohol and ether. They are stained in concave 
watch-crystals, specimen side down, so that the precipitate which is 
invariably formed during the process of staining does not adhere too 
firmly to the smeared surface. After from seven to ten minutes the 
preparations are thoroughly washed with water and examined in a 
drop of water before making mounts in balsam, in order to ascertain 
whether the staining has been successful, especially in so far as the 
malarial organisms are concerned. If the nuclei of the large mono- 
nuclear leucocytes are colored dark red, it may generally be inferred 
that the parasites also will be properly stained. In that event the 
cover-glass is carefully removed from the slide, dried in the air, and 
mounted in balsam. The latter must be free from acid or nearly so, 
as otherwise the characteristic coloring is lost at once. 

An advantage of the method is that specimens older than three or 
four weeks may still be satisfactorily examined. In that event, 
however, it is necessary to place them in alcohol for some time and 
then to wash in water so as to be sure that they will actually be 
moistened by the watery solution of the dye. In old specimens it is 
also necessary to stain for a much longer time — often for twenty-four 
hours. Should the preliminary examination in water show that the 
red cells are stained dark blue or a dark bluish red, which is com- 
mon with specimens that are old, it is well to dip the specimen into 
alcohol for a second or two so as to extract some of the dye ; it is 
then again washed in water, dried, and mounted. 

In a properly-stained Romanowsky specimen, no matter what modi- 
fication has been used, the red cells appear red, in overstained or old 
specimens light gray or light blue. Polychromatophilia and granular 
degeneration are well shown. The neutrophilic granules are bright 
red, the eosinophilic granules eosin colored, and the mast-cell gran- 
ules dark red. The nuclei of the lymphocytes, large mononuclear 
leucocytes, and myelocytes are bright red, those of the polynuclear 
leucocytes a bluish violet. In some of the lymphocytes and large 
mononuclear leucocytes Michaelis' granules will be seen. The blood- 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 129 

plates are pale blue with red nuclei. The nuclei of the red blood- 
corpuscles are red. The malarial organisms present a blue body 
with one or more intensely red nuclear structures varying in size 
from that of a tiny dot in the youngest forms to a structure which 
in the microgametocytes fills the entire body of the parasite in the 
form of a fine reticulum. In the segmenting bodies it will be 
observed that each segment contains a red nucleus, while the body 
is blue. 

Method of Michaelis and Wolff. 1 — Two solutions are kept 
on hand : the one is a 1 pro mille aqueous solution of eosin ; the 
other is a methylene-azure solution, which is prepared as follows : 
200 c.c. of a 1 per cent, aqueous solution of methylene-blue are 
boiled for fifteen minutes with 10 c.c. of a decinormal solution of 
sodium hydrate and then accurately neutralized with 10 c.c. of a 
decinormal solution of sulphuric acid. Immediately before use, 2 
c.c. of the resulting azure blue solution are well mixed with 10 c.c. 
of the eosin solution. The preparations, which are best fixed by 
immersion for one hour in absolute alcohol, are placed in watch- 
crystals in the staining solution, specimen side down, and are 
allowed to remain for fifteen minutes. They are then washed in 
water, dried, and mounted as described above (see Xocht's method). 
The occurrence of a precipitate during the process of staining is 
disregarded. The various elements of the blood are stained as with 
Nocht's method. 

Method of Giemsa. 2 — Giemsa uses a 1 pro mille aqueous solu- 
tion of eosin (yellow shade) and a 1 pro mille aqueous solution of 
pure azure blue (Grubler). Before use, 1 c.c. of the eosin solution 
is diluted with 10 c.c. of water and treated with 1 c.c. of the 
azure solution. The specimens are fixed by heat or by immersion 
in absolute alcohol for one hour, and are then placed in the staining 
solution, specimen side down, using watch-crystals, for a time varying 
between ten minutes and one hour, which must be ascertained by 
trial. They are then washed in water, dried, and mounted as above ; 
the individual elements are stained as with Nocht's method. 

Method of Reitter. 3 — Reuter precipitates a solution of methyl- 
ene-azure in methylene-blue prepared according to Michaelis or 
Nocht, with a dilute solution of eosin, and makes use of the pre- 
cipitated eosinate of methylene-blue mixed with eosinate of methyl- 
ene-azure in alcoholic solution (best methyl alcohol to which some 
anilin oil is added). A few drops of this solution are added to a 
watch-cry stalful of water, in which the specimens are immersed for 
one hour. They are then washed in water, dried, and mounted as 
usual. Precipitates are not formed during the process of staining, 

1 L. Michaelis u. A. Wolff, Virchow's Archiv, 1902, vol. clxvii. p. 151. 

2 Giemsa, Centralbl. f. Bakt., 1902, vol. xxxi. 

3 Eeuter, Ibid., 1901, vol. xxv. 



130 THE BLOOD. 

but as I have stated above, even the alcoholic solution loses much 
of its staining power after a few weeks. 

The dye can be procured from Griibler already prepared. 

The individual elements of the blood are stained as with Nocht's 
method (see above). 

Leishman's Method. 1 — Leishman also makes use of the isolated 
eosinate of methylene-blue mixed with eosinate of methylene-azure. 
He proceeds as follows : Two solutions are prepared : one a 1 pro 
mille solution of eosin (Griibler' s extra B.A.) in distilled water ; the 
other a 1 per cent, solution of medicinal methylene-blue (Griibler), 
also in distilled water, and alkaliuized with sodium carbonate to the 
extent of 0.5 per cent. This last solution is heated to 65° C. for 
twelve hours, and is then allowed to stand at the temperature of the 
room for ten days before using. Equal volumes of the two solutions 
are mixed in a large open basin and are allowed to stand for from 
six to twelve hours, the mixture being stirred from time to time with 
a glass rod. The resulting precipitate is collected on a filter, 
thoroughly washed with distilled water, dried, and powdered. A 
0.15 per cent, solution of the dye in pure methyl alcohol serves as 
stain and does not deteriorate on keeping. Special fixation is not 
required. The blood-film is covered with the solution and stained 
for about one-half minute. Double the amount of distilled water 
is then added and allowed to mix with the alcoholic solution. After 
from five to ten minutes the stain is washed off with distilled water, 
a few drops of water being allowed to rest on the film for a minute. 
The specimen is next dried (without heat) and can then be examined 
as usual. The soaking in water for a minute after the staining is 
important, as it intensifies the Romanowsky stain ; it changes the 
tint of the red corpuscles from a greenish-blue to a transparent pink 
or greenish color, while the nuclei of the leucocytes are usually a 
ruby red. The nuclei of nucleated red cells are almost black and 
the extranuclear portion gray. The blood-plates are a deep ruby 
red with shaggy margins, frequently showing a pale-blue peripheral 
zone surrounding the red centre. The body of the malarial parasite 
stains blue and its chromatin a ruby red. In the case of the tertian 
parasite SchmTner's dots are well marked in the containing red 
corpuscles. 

Wright's Modification of Leishman's Method. 2 — Wright 
has simplified Leishman' s method in several important particulars, 
which render the method even more convenient for routine work • 
he has ascertained, moreover, that any one of the Griibler methylene- 
blues can be employed for the purpose of obtaining a sufficient 
quantity of methylene-azure. 

The staining fluid is prepared as follows : 1 per cent, of methylene- 

1 Leishman, Brit. Med. Jour., Sept. 21, 1901. 

2 Wright, Jour. Med. Kesearch, 1902, vol. vii. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 131 

blue is added to a 1 per cent, aqueous solution of sodium bicarbonate, 
when the mixture is steamed in an Arnold's steam sterilizer for one 
hour. On cooling, the solution is poured directly into a large dish or 
flask and treated, while stirring or shaking, with a sufficient quantity 
of a 1 pro mille solution of eosin (yellow shade) until the mixture 
has assumed a purple color and a scum with a metallic lustre forms 
on the surface. This will require about 500 c.c. of the eosin solu- 
tion for 100 c.c. of the methylene-blue solution. The resultant 
precipitate, which contains both eosinate of methylene-blue and 
eosinate of methylene-azure, is collected on a filter, and without 
further washing is allowed to dry. When thoroughly dry, an 0.3 
per cent, solution in pure methyl alcohol is prepared (this is practi- 
cally a saturated solution). The solution is then filtered and to the 
filtrate 25 per cent, methyl alcohol is further added so as to dilute 
the stain somewhat and to lessen the tendency of the dye to become 
precipitated during the process of staining. 

The air-dry blood -films are covered with the stain for one minute ; 
water is then added drop by drop until the staining fluid becomes 
semitranslucent and a reddish tint becomes visible at the margins, 
while a scum with a metallic lustre forms on the surface. The 
amount of water required will vary with the amount of staining 
fluid on the preparation, but in general it may be said that 8 or 10 
drops will suffice if a seven-eighths inch square cover-glass is used. 
The staining fluid, thus diluted, is allowed to remain on the prepara- 
tion for two or three minutes, during which time the real staining 
of the specimen takes place. It is then washed off, when the blood- 
film will be seen to have a blue or purple color. 

The next step is to develop the differential staining of the various 
elements in the preparation. This is done by washing the prepara- 
tion in water, preferably distilled water, until the better spread 
portions of the film appear yellowish or reddish in color. If desired, 
the process of differentiation may be readily observed by placing the 
cover-glass, film side uppermost, on a slide, covering it with water, 
and examining it with the microscope under a low magnifying power. 
The red blood-corpuscles, which, as before stated, at first have a blue 
color, will become greenish, then yellowish, and finally orange or 
pinkish in color, depending upon the depth of the original staining, 
which varies with the length of time that the diluted staining fluid 
has been allowed to act, and with the degree of its dilution. 

The differentiation by washing in water seems to be essentially a 
process of decolorization by which some of the blue constituent of 
the dye is removed, for the water that drains off from the prepara- 
tion has a blue color. This differentiation or decolorization proceeds 
slowly, and may require from one to three minutes, depending upon 
the intensity of the staining and upon the tint sought to be obtained 
in the red corpuscles. 



132 THE BLOOD. 

It is apparent from the above that with a little experience with 
the method the color of the red corpuscles may be made either orange 
or pink. When the desired color is obtained in the red corpuscles, 
the preparation is quickly dried between layers of filter-paper and 
mounted in balsam. It is important to arrest the decolorization by 
drying the preparation as soon as the desired tint in the red corpuscles 
is obtained, for it may be carried too far. 

Dried blood-films may be kept for weeks without impairment of 
their staining properties. Films months old will probably not give 
good results. 

In a suitably stained specimen the red cells are either orange or 
pink ; polychromatophilia and granular degeneration are well shown 
(the granules blue) ; the neutrophilic granules are a reddish lilac, 
the eosinophilic granules eosin colored, the mast-cell granules a dark 
blue, a dark purple, or even black. The lymphocytes have dark 
purplish-blue nuclei with robin's egg blue protoplasm, in which the 
granules described by Michaelis appear dark blue or purplish. The 
large mononuclear leucocytes present a blue or dark lilac colored 
nucleus, and in some Michaelis' granules can also be made out. 
The blood-plates are stained a deep blue or purplish and the malarial 
organisms are colored as with Nocht's method. 

Koch's Method. 1 — With Koch's method the modification of the 
methylene-blue with an alkali is effected during the process of 
staining. Three stock solutions are kept on hand : the one is a 
concentrated aqueous solution of medicinal methylene-blue (Hochst) ; 
the other is a 1 per cent, aqueous solution of eosin (Hochst B. A. 
extra) ; the third is a 5 per cent, aqueous solution of crystallized 
sodium carbonate. Before use, the different components are mixed 
as follows : 1 c.c. of the methylene-blue solution is diluted with 10 
c.c. of distilled water ; to this 3 drops of the soda solution are 
added, and, while stirring, as much of the eosin solution as will 
produce a finely granular precipitate. 

The blood-films should be fixed by heat or with alcohol and ether, 
and are stained from five to ten minutes, when they are differentiated 
in distilled water (about 10 c.c.) to which an oeseful of acetic acid 
has been added. The differentiation is carried to a point where the 
eosin tone becomes apparent. The specimens are then rapidly washed 
off, dried, and mounted as usual. 

The various elements will be stained as above. 

Ewing's Method. 2 — Ewing makes use of a neutralized solution 
of Unna's polychrome methylene-blue, which is itself rich in methyl- 
ene-azure, and which he then combines with eosin and a small 
amount of methylene-blue. His method in detail is the following : 

A neutral solution of Unna's polychrome methylene-blue is pre- 

1 Koch, Dentsch. med. Woch., 1900. 

2 Ewing, Clinical Pathology of the Blood, loc. cit. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 133 

pared by adding dilute acetic acid (2-3 per cent, solution) to the 
commercial polychrome methylene-blue solution (Griibler), until the 
latter no longer presents an alkaline reaction. As a general rule 5 
drops of a 3 per cent, solution of the acid are sufficient for 1 ounce 
of the commercial liquid dye. The reaction is tested with red 
litmus-paper, taking note of the color immediately above the zone 
which comes in contact with the stain. 

In addition to the above solution Ewing uses a 1 per cent, 
aqueous solution of Griibler' s methylene-blue, at least a week old, 
and a 1 per cent, aqueous solution of Griibler's eosin (yellow shade). 

The staining reagent is then prepared by adding 4 drops of the 
eosin, 6 drops of the polychrome methylene-blue, and 2 drops of the 
ordinary methylene-blue solution to 10 c.c. of distilled water and 
mixing well. The blood specimens are fixed in alcohol or by heat 
and are immersed in the stain, specimen side down, for one or two 
hours, or if necessary even longer ; they will not overstain in twenty- 
four hours. They are then washed in water, dried, and mounted as 
usual. The different elements of the blood will be stained as with 
JNocht's method. 

Ziemann's Method. 1 — This is warmly recommended by Grawitz 
as a routine laboratory method. Two stock solutions are employed, 
the one a 1 pro mille aqueous solution of eosin, the other a 1 per 
cent, aqueous solution of methylene-blue containing 2.5 grammes of 
borax in 100 c.c. Before use, the two solutions are mixed in the 
proportion of 4 parts of the eosin to 1 of the methylene-blue solution 
and poured over the fixed blood-smears lying face downward in 
watch-crystals with concave bottom. After five minutes the speci- 
mens are washed in water, the iridescent film which forms being thus 
removed. At this stage the preparations are colored an intense 
bluish violet, which changes to a reddish tint when they are im- 
mersed for a moment or two in very dilute acetic acid solution. 
They are then again washed in water, dried, and mounted. The 
various elements of the blood are stained as already described (see 
Nocht's method). 

Ehrlich's Eosin-methylene-blue-methylal Solution. 2 — In this method 
the neutral dye which is formed from the eosin and methylene-blue 
is held in solution with methylal (methylendimethylether). The 
reagent consists of 10 c.c. of a 1 per cent, aqueous solution of eosin, 
to which 8 c.c. of methylal and 10 c.c. of a saturated aqueous solu- 
tion of medicinal methylene-blue are added. The mixture is ready 
for use at once, and furnishes good results ; it is very unstable, how- 
ever, and should be freshly prepared whenever it is needed. It is 
necessary that the specimens should be carefully fixed by heat, as a 
characteristic coloring is otherwise not obtained. The preparations 

1 As described by Grawitz, Pathol, d. Blutes, 2d ed, 

2 Ehrlich-Lazarus, Die Anaemie, 1898. 



134 THE BLOOD. 

are stained for one or two minutes. The eosinophilic granules are 
stained red, the mast-cell granules a pure blue, and the neutrophilic 
granules a purplish red. 

In my experience the method presents no advantages over Jen- 
ner ? s method and is decidedly more complicated. 

Michaelis' Eosin-methylene-blue-acetone Solution. 1 — The neutral dye 
resulting from the interaction between the eosin and the methylene- 
blue is here held in solution by the aid of acetone. Like the 
preceding method, it has no advantages over Jenner's method, 
and is decidedly more complicated. Two solutions are prepared, 
viz., one containing 20 c.c. of a 1 per cent, aqueous solution of 
chemically pure methylene-blue with an equal amount of absolute 
alcohol ; the other is composed of 1 2 c.c. of a 1 per cent, aqueous 
solution of chemically pure eosin and 28 c.c. of acetone. These two 
solutions are kept in separate bottles and are mixed in equal propor- 
tions immediately before use. The mixture is placed in a watch- 
crystal and is covered at once. The blood-films should be fixed by 
heat or by immersion in absolute alcohol for from one to twenty- 
four hours, and are then placed in the stain, face downward, for 
from one-half to ten minutes, the time varying with the individual 
preparations. The staining should be interrupted as soon as the 
blue color which first appears has turned to red, as otherwise the 
nuclei of the leucocytes will be decolorized. If the leucocytes are 
materially increased, it is best to stop even before this point is 
reached. If, on the other hand, the blue stain has acted too ener- 
getically, the specimens are stained a little longer. The preparations 
are finally rinsed in water, thoroughly dried, and mounted as usual. 
The various elements of the blood in a successfully stained specimen 
will be found colored as with Jenner's method. 

Strauss and Rohnstein's Method. 2 — The preparations are first 
stained with a solution of rubeosin and then counterstained with 
methylene-blue, which stains in an elective manner. Strauss and 
Eohnstein suggest the method for routine' work. The technique in 
detail is the following : The blood-smears are fixed by heat, alcohol- 
ether, or formalin, and after drying are stained for three minutes in 
a mixture of the following solutions (3 parts of A to 1 part of B) : 

A 

Eosin (yellow shade, Griibler) 0.5 gramme 

Absolute alcohol 80.0 grammes 

Water 20.0 

B 

Kubin (fuchsin, Griibler) . 0.5 gramme 

Absolute alcohol 80.0 grammes 

Water 20.0 

' i Michaelis, Deutsch. med. Woch., 1899, p. 490, and 1901. 

2 H. Strauss u. B. Eohnstein, Die Blutzusammensetzung b. d. verschiedenen 
Anaemien, Berlin, 1901, Hirschwald. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 135 

The specimens are then washed in water, stained in a ^ to -J per cent, 
aqueous solution of the methylene-blue (which if possible should be 
at least a couple of weeks old), again washed in water, dried, and 
mounted. 

The red cells are colored a brick red, the nuclei and all basophilic 
elements marine-blue, the neutrophilic granules a bluish-brownish 
violet, and the eosinophilic granules a bright red. 

Strauss and Rohnstein state that overexposure in one or the other 
solution does not matter very materially, and that the AB mixture 
keeps for a long time. 

Special Staining" Methods. — Staining with Eosin. — The blood- 
smears are best fixed by heat or by immersion in absolute alcohol 
and are then stained for about a minute with an 0.25 to 0.5 per 
cent, alcoholic (70 per cent.) solution of eosin, or for ten to twenty 
minutes with an 0.1 to 0.5 per cent, aqueous solution of the dye. 
They are then washed in water, dried, and mounted as usual. The 
red corpuscles and the eosinophilic granules are stained a bright red, 
and the protoplasm of the leucocytes a faint red. 

The stain is used to demonstrate the presence of oxyphilic 
material. 

Staining with Ehrlich's Triglycerin Mixture. 1 — This is composed 
of 2 grammes each of eosin, aurantia, and indulin in 30 grammes 
of glycerin. These constituents are brought into solution by keep- 
ing the mixture in the warm chamber (37° to 40° C.) for about 
one week. 

The specimens must be well fixed, an exposure to a temperature 
of about 110° C. for at least two hours being best. They are then 
allowed to remain upon the stain for from sixteen to twenty-four 
hours, when they are rinsed in water, dried, and mounted as usual. 
The red corpuscles are colored orange, the bodies of the leucocytes a 
dirty gray, with dark nuclei, and the eosinophilic granules a bright 
red. If, however, the specimens are fixed too intensely, the eosino- 
philic granules take the aurantia and are thus colored orange. 

The stain is used for the demonstration of oxyphilia and its degree. 

Ehrlich's Neutral Mixture. 2 — This consists of 5 volumes of a 
saturated aqueous solution of acid fuchsin, to which 1 volume 
of a saturated aqueous solution of methylene-blue is slowly added 
while stirring. The mixture is treated with 5 volumes of distilled 
Avater and filtered after having stood for several days. The speci- 
mens are stained for from five to twenty minutes. Only a slight 
degree of fixation is necessary. 

The red corpuscles are stained the color of fuchsin, their nuclei as 
well as those of the leucocytes are black or a light lilac, the eosino- 
philic granules red, and the neutrophilic granules violet. 

The stain is used for the demonstration of neutrophilic material. 

1 Ehrlich-Lazarus, loc. cit. 2 Ibid. 



136 THE BLOOD. 

Basic Double Staining. — Ehrlich's Methyl-green-fuchsin Mixt- 
ure. 1 — A saturated aqueous solution of methyl-green is treated 
with a small amount of an alcoholic solution of fuchsin. After brief 
fixation the specimens are stained for five minutes. The nuclei 
appear green, the red corpuscles red, and the protoplasm of the 
lymphocytes the color of fuchsin. The stain is especially service- 
able for demonstration purposes in cases of lymphatic leukaemia. 

Instead of methyl-green, one can also use iodine-green, but not 
malachite-green or chromgreen ; instead of the fuchsin, para- (rosani- 
lin) rubin, saifranin, acridin-red, neutral red, or best of all pyronin 
may be employed. 

Pappenheim's Method. 2 — Pappenheim suggests a stain which is 
composed of a concentrated aqueous solution of methyl-green, to 
which pyronin is added until the solution just begins to turn 
blue viz., about 1 part of pyronin for 3 to 4 parts of methyl-green. 
Stained in this manner the basophilic protoplasm of the lympho- 
cytes is colored a fine dark carmine red, while the protoplasm 
of all other cells is stained a more or less pale brownish or reddish- 
yellow, or remains colorless. Pappenheim regards this stain as 
essentially specific for the lymphocytes, but admits that it also stains 
in a similar maimer the young erythroblasts that are poor in haemo- 
globin. The difference can be recognized from the character of the 
nuclei and the fact that the margin of the lymphocytes very com- 
monly appears shaggy, while that of the erythroblasts is smooth and 
homogeneous. 

Special Mast-cell Stains. — Ehrlich's Dahlia. 3 — The staining fluid 
consists of 100 c.c. of distilled water to which 50 c.c. of a saturated 
alcoholic (absolute) solution of dahlia are added. On clearing, this 
solution is further treated with 10 to 12.5 c.c. of glacial acetic acid. 
The blood-smears should be fixed by heat or absolute alcohol, and 
are then stained for from five to ten minutes. With the exception 
of bacteria, only the mast-cells are stained, while the neutrophilic 
leucocytes are only faintly tinged. 

Pure methyl-green does not stain the mast-cell granules, but the 
impure products which are contaminated with methyl-violet give 
good results. This is owing to the fact that methyl-green is a pure 
nuclear dye. 

Westphal's Method. 4 — This furnishes very handsome pictures. 
100 c.c. of a carmine solution are prepared by dissolving 2 grammes 
of pure carmine in 200 c.c. of distilled water with 5 grammes of 
alum ; the mixture is boiled for fifteen minutes, filtered, and treated 
with 1 gramme of carbolic acid. This solution is then mixed with 
100 c.c. of glycerin, 100 c.c. of a concentrated solution of dahlia in 

1 Ehrlich-Lazarus, loc. cit. 

2 Pappenheim, Virchow's Archiv, 1899, vol. clvii. 

3 Ehrlich-Lazarus, loc. cit. 

4 Westphal, Inaug. Diss., Berlin, 1880. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 137 

absolute alcohol, and 20 c.c. of glacial acetic acid. The specimens 
are stained for about twenty-four hours. The mast-cell granules 
and any bacteria that may be present are colored a bluish violet, 
while all nuclei are colored red. 

Methylene-azure. — Methylene-azure is also well adapted for the 
demonstration of mast-cells ; it may be used in any one of the modi- 
fications described under the heading of the Romanowsky stain. The 
granules are stained metachromatically (see page 80). 

Methods of Chenzinsky -Plehn. — The methods of Chenzinsky and 
Plehn, 1 which were formerly extensively used in the study of ma- 
larial blood, are now rarely employed. The staining reagents are 
eosin-methylene-blue mixtures, in which the resulting neutral dye, 
the eosinate of methylene-blue, is held in solution by an excess of 
the basic component. Both methods are here described, as they 
may possibly still be used when other reagents are not available. 

Chenzinsky Method. — The reagent consists of 40 c.c. of a concen- 
trated aqueous solution of methylene-blue, to which 20 c.c. of a 0.5 
per cent, solution of eosin in 70 per cent, alcohol and 40 c.c. of 
distilled water have been added. The solution keeps fairly well, 
but should always be filtered before using. A slight degree of fixa- 
tion only is necessary — five minutes in absolute alcohol. The speci- 
mens are stained for from six to twenty-four hours in air-tight 
watch-crystals at a temperature of 37° to 40° C. 

The red corpuscles and eosinophilic granules are stained a bright 
red, the nuclei and mast-cell granules a dark blue, the bodies of the 
malarial organisms a light sky blue. The neutrophilic granulation 
remains unstained. 

Plehn's Method. — The stain consists of 60 c.c. of a concentrated 
aqueous solution of methylene-blue, 20 c.c. of a 0.5 per cent, solu- 
tion of eosin in 75 per cent, alcohol, 40 c.c. of distilled water, and 
1 2 drops of a 20 per cent, solution of caustic alkali. 

The specimens are fixed by heat and stained as with Chenzinsky' s 
method ; the coloring is the same. 

Futcher's Carbol-thionin. — This method, which was formerly ex- 
tensively employed in the study of malarial blood, is no longer in 
common use. It gives good results, but unfortunately the specimens 
soon fade out. 

The air-dried films are fixed for one minute in a 0.25 per cent, 
solution of formalin in 95 per cent, alcohol. But as it is important 
that this solution should be made up fresh for each examination, it 
is more convenient to keep a 10 per cent, aqueous solution of forma- 
lin on hand, and to add 4 or 5 drops of this to 10 c.c. of 95 per 
cent, alcohol just before using. The specimens are then rinsed in 
water, dried between filter-paper, and stained for from ten to fifteen 
seconds with a carbolated solution of thionin. This is prepared by 

1 Plehn, Aetiol. u. klin. Malariastud., Berlin, 1890, Hirschwald. 



138 THE BLOOD. 

adding 20 c.c. of a saturated solution of thionin in 50 per cent, 
alcohol to 100 c.c. of a 2 per cent, solution of carbolic acid. The 
tbionin carbolate tbus formed constitutes the active staining prin- 
ciple. After washing off the excess of stain the preparations are 
dried with filter-paper and mounted as usual. Thus prepared, the 
malarial parasites appear as reddish-violet bodies and are readily 
seen. The method is of special value in staining the ring-shaped 
bodies of the sestivo-autunmal infection, which are difficult to recog- 
nize in unstained specimens, and usually do not stain well with 
eosin and methylene-blue. 

Staining with Ehrlich's Hematoxylin- eosin, or Orange-G 
Solution. — The solution is prepared by dissolving 2 grammes of 
hematoxylin in a mixture of 100 grammes each of distilled water, 
alcohol, and glycerin. To this solution 10 grammes of glacial 
acetic acid and an excess of alum are added. After exposure to the 
sunlight for from four to six weeks about 0.5 gramme of eosin or 
orange-G is added. 

The specimens are fixed in absolute alcohol, or by heat (a brief 
exposure only is necessary). They are then left in the stain, in the 
sunlight, for from one-half to two hours, when they are thoroughly 
washed in water, dried, and mounted. 

The red corpuscles and eosinophilic granules are colored a bright 
red, the nuclei of normoblasts and megaloblasts a deep black, the 
bodies of the leucocytes a light lilac, and their nuclei a dark lilac. 
The bodies of the lymphocytes, however, are scarcely stained at all, 
while their nuclei appear only a shade lighter than those of the 
nucleated red corpuscles. 

Demonstration of Iodophilia. — Cover-glass specimens are pre- 
pared as usual ; after drying in the air they are placed in a small jar 
containing a few crystals of iodine. After several minutes the films 
assume a dark-brown color, when they are mounted in a drop of a 
saturated solution of laevulose and examined with an oil-immersion 
lens. The red corpuscles are stained the color of iodine, while the 
leucocytes are almost colorless. All glycogen granules, whether 
contained in leucocytes or free in the blood, are stained a distinct 
mahogany. 

This method furnishes better results than the older method of 
staining with a solution composed of 1 gramme of iodine and 3 
grammes of potassium iodide in 100 grammes of a concentrated 
solution of mucilage (1 part of LugoFs solution to 100 parts of 
a thick mucilage). 1 

In place of the method just described, Barfurth's procedure may 
also be employed. In this case the solution is prepared by mixing 
1 part of Lugol's solution with 2 parts of glycerin. The prepara- 
tions do not keep well, however, as the glycerin gradually extracts 
the iodine. 

1 Ehrlich-Lazarus, Die Anaemie, loc. cit. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 139 

Distribution of the Alkali in the Blood. 

A very good idea of the distribution of the alkali in the blood 
may be formed by making use of the following method, suggested 
by Ehrlich : a drop of blood is carefully spread between two cover- 
glasses, when the air-dried specimens are immediately placed in a 
watch-crystal, containing a solution of the free staining acid of ery- 
throsin in chloroform. In a few minutes the specimens have 
assumed a bright-red color, when they are transferred for a minute or 
two into a crystal containing chloroform. While still moist they 
are then imbedded in Canada balsam. Prepared in this manner, 
the alkaline elements of the blood are colored red. The plasma 
presents a distinctly red color, while the red corpuscles have not 
taken up the stain. The protoplasm of the leucocytes and especially 
of the lymphocytes, as also the plaques, the fibrin-filaments, and the 
bits of protoplasm derived from the leucocytes are all stained a deep 
red, while the nuclei of the leucocytes remain colorless. If mala- 
rial organisms are present, these are likewise stained. 

In order to prepare the stain, a saturated aqueous solution of 
erythrosin (tetra-iodo-fluorescein) is acidified with dilute hydrochloric 
acid, and the staining acid, which is thus precipitated, collected on a 
filter, after having been washed with distilled water. The precipi- 
tate is dissolved in chloroform, to which it imparts an orange color. 
This solution is employed for staining. In every case care should 
be had that the glass utensils which are used are freed from adherent 
alkali by washing with concentrated acids and then with distilled 
water. 

Enumeration of the Corpuscles of the Blood. 

Method of Thoma-Zeiss. — Of the various instruments devised 
for the enumeration of the corpuscles of the blood, that of Thoma- 
Zeiss is almost exclusively used in the United States. Its essential 
parts are two diluting pipettes and a counting-chamber. One 
pipette permits of a dilution of the blood in the proportion of 1 : 100 
to 1 : 1000, and is generally used in the enumeration of the red cor- 
puscles ; the other pipette, which has been devised for counting the 
white cells, will allow a dilution of 1 : 10 to 1 : 100. Each pipette 
has a small bulb blown on its stem, which contains a glass bead to 
facilitate the mixing of the blood and the diluting fluid. The prin- 
ciple underlying the construction of the counting-chamber will be 
seen in the accompanying illustration (Fig. 17). It is essentially a 
large slide to which the glass plate D is cemented. This, it will be 
noted, has its central area cut out, and there is cemented to the centre 
of this area a round platelet of glass, the surface of which is exactly 
0.1 mm. below the surface of I). If then a cover-glass is placed 
upon D, there will be an underlying chamber, B, which is filled by 



140 



THE BLOOD. 



a droplet of the diluted blood. To facilitate enumeration of the 
individual corpuscles, a portion of the central platform is ruled into 
sets of small squares, each of which has an area of exactly -^\-^ 
square mm. As the depth of the chamber is 0.1 mm., each square 
represents the base of a cube, the contents of which Avill be T ^ X 
y 1 ^ — that is, y^Vo c ^ mm « I n counting the red cells the object is to 
ascertain the average number of cells in a small square by counting 
a large number of squares, when the corresponding number in the 
undiluted blood is ascertained by multiplying by 4000 and the degree 



Fig. 17. 





0.100 mm. 
im mm. 


© 








Thoma-Zeiss blood-counting apparatus. 

of dilution. Similar considerations underlie the determination of the 
number of leucocytes per unit of blood — i. <?., per cbmm. (see below). 

In the accompanying diagram (Plate IX.) I have represented the 
Turk ruling which is especially convenient. It is a modification of 
the Zappert ruling and well adapted for counting the leucocytes and 
red corpuscles in one and the same specimen. It is made up of large 
and small squares, which for convenience* sake I have represented 
partly in black and partly in red. 

Special Technique. — Enumeration of the Red Cells. — To secure 
the necessary amount of blood, it is more convenient to puncture 
the finger than to obtain the blood from the ear, as the hand of the 
operator can be steadied against the finger of the patient. The 
finger is washed with soap and water, then with alcohol and dried. 
The puncture should be sufficiently deep that a large drop of 
blood can be obtained without special pressure. If there is reason to 
believe that the patient is not very anaemic, the capillary pipette is 
filled to the mark 0.5, which will furnish a dilution of 1 : 200 ; 
otherwise the blood is drawn to the mark 1, which will give a dilu- 
tion of 1 : 100. In filling the pipette the tube is held between the 
thumb and forefinger of the right hand, while the finger of the patient 



PLATE IX. 

































































■ 










ip 










— 


==== - 























iiEfi 






. 








. L-I--J. 















li 1 


1 1 i 


— H 

U- 









Counting Chamber. 



Turk': 

are in an I44 ,ar g e scares, for — jS^E-iS ^ "■* 

ruled in red, is used in counuus 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 141 

is held with the fingers of the left hand ; at the same time the right 
hand is steadied by placing the little finger or ring finger of the right 
hand against the patient's finger from which the blood is being drawn. 

As soon as the requisite amount of blood has been obtained, the 
end of the pipette is rapidly but carefully wiped of adherent blood 
with the finger and then immediately plunged into the diluting fluid, 
which should be ready at hand. This is drawn up to the mark 101, 
while the pipette is gently rotated between two fingers. When the 
101 mark has been reached, the blood is intimately mixed with 
the diluting fluid by further rotation. The contents of the capil- 
lary tube are now expelled, the remaining mixture representing a 
1 : 100 or 1 : 200 dilution of the blood, as the case may be. 

A few precautions during this step of the process should be 
observed : It is essential that the degree of dilution should be 
accurate, and it is therefore a prime requisite that the blood column 
should just reach the 0.5 or the 1 mark, as the case may be, and 
that similarly the 101 line is not exceeded. If the blood has been 
drawn too far, as often happens with beginners, the tube must be 
cleansed and another attempt made. In such a case the diluting 
fluid is drawn up exactly as though no accident had happened, 
so as to prevent the column of blood from coagulating in the capil- 
lary tube. In any event only a few seconds should be allowed to 
elapse from the time that the tube is filled with the blood until the 
diluting fluid is drawn up. Should air-bubbles enter the blood 
column, the entire process must, of course, be repeated. 

For the purpose of diluting the blood Toison's solution is most 
commonly used. It has the following composition : 

Sodium chloride 1.000 gramme. 

Sodium sulphate 8.000 grammes. 

Neutral glycerin 30.000 " 

Distilled water 160.000 " 

Methyl-violet 5 B 0.025 gramme. 

The addition of the methyl-violet serves the purpose of coloring 
the leucocytes, which are thus rendered quite easily visible. 

Other solutions which may be employed are the following : phys- 
iological salt solution — viz., 0.9 per cent. ; a 15—20 per cent, solu- 
tion of magnesium sulphate ; a 5 per cent, solution of sodium 
sulphate ; one of osmic acid (1 : 4000) ; Lugol's solution (iodine, 
1 gramme ; potassium iodide, 2 grammes ; and water, 300 c.c.) ; 
the special reagents of Hayem, Pacini, Petrone, Acquisto, and Mar- 
cano, the formulae of which follow : 

Formula of Hayem' s fluid : 

Mercuric chloride 0.5 gramme. 

Sodium sulphate 5.0 grammes. 

Sodium chloride 2.0 " 

Distilled water . 200.0 " 



142 THE BLOOD. 

Formula of Pacini's fluid : 

Mercuric chloride 2.0 grammes. 

Sodium chloride . 4.0 " 

Glycerin 26.0 

Distilled water 226.0 

Formula of Petrone's fluid : 

Mercuric chloride 1.0 gramme. 

Sodium chloride 7.0 grammes. 

Distilled water ' . . 100.0 

Of this solution 10 drops are diluted with 90 drops of distilled 
water ; the resultant solution is used as the diluent. 

Formula of Acquisto's solution : 

An 0.5 per cent., solution of chromic acid 1 part. 

Sulphopicric acid 1 " 

A 1 pro mille solution of mercuric chloride 1 " 

A mixture of absolute alcohol and \ glacial acetic acid . 1 " 

The resultant solution is diluted with 2 parts of water before use. 

Formula of Marcano's reagent : 

Formol 1.0 gramme. 

Sodium chloride 1 .0 " 

Distilled water . 85-100 grammes. 

Before use, the liquid is diluted with 2 volumes of distilled water. 

After the pipette has been filled in the manner indicated and the 
contents of the capillary tube have been expelled, a drop of the 
diluted blood is placed on the platform of the counter and is cov- 
ered with one of the specially ground cover-glasses which accom- 
pany the instrument. This step of the process requires great care 
and some experience. It is essential that the apparatus should 
be perfectly clean and dry, that no air-bubbles enter, that the entire 
platform is covered, and that none of the liquid flows over between 
the cover-glasses and the plate D. It is necessary, moreover, that 
the corpuscles should be evenly distributed over the platform, which 
is readily ascertained by a superficial examination with a low power 
after the corpuscles have settled. In a well -prepared specimen New- 
ton's colored rings will appear between the cover-glass and the plate 
D, if moderate pressure is exerted upon the cover, especially if the 
surface of D has been slightly moistened by breathing upon it 
very gently before the cover-glass is applied. Personally I advise 
that the drop should be of such size that it exactly covers the plat- 
form after the cover is adjusted, and that no portion of the liquid 
shall project over or flow into the moat. Otherwise currents are set 
up which are especially apt to produce an irregular distribution of 
the cells. The cover-glass must be rapidly adjusted and firmly 
pressed into position. If then a rapid survey shows that the speci- 
men is satisfactory, the count is begun. Using a Leitz No. 7 or 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 143 

a Bausch & Lomb J, 1 the left upper large square, composed of 16 
small squares, is brought into the field (see Plate IX.). In this the 
red cells are counted in rows of 4 horizontal small squares. The 
slide is then moved to the adjoining large square. In doing so it 
will be noted that every large square is separated from its neighbor 
both horizontally and vertically by a row of small cells traversed 
by a mesially placed line, which serves as a guide to the next large 
square. As a general rule it will be found more convenient to 
ignore these intermediary squares and to take account only of the 
large ones. The results are marked as in the first square, and so on 
throughout the entire set of 16 large squares, cells that lie on the 
boundary-line of a square being included when they touch the upper 
and left border, while they are excluded when they are found on the 
right and lower lines. The final calculation is made as follows : 
All the red cells counted are summed up and divided by the corre- 
sponding number of small squares, so as to ascertain the average 
number of red cells for 1 small square, the cubic contents of which, 
as we have seen, is 40 1 cbmm. To ascertain the number of red 
cells in 1 cbmm. of diluted blood, this number is hence multiplied by 
4000, and the result by the degree of dilution, which will give the 
number of red cells in 1 cbmm. of undiluted blood. 

Example. — Dilution, 200 times ; number of red cells counted in 
the 16 large squares—^, e., 16X16=256 small squares — was 1536; 
this means 6 cells for each small square or -£-$-$-$ cbmm. ; hence in 1 
cbmm. of diluted blood 6 X 4000 = 24,000 cells, and in 1 cbmm. 
of undiluted blood 24,000 X 200 = 4,800,000 red corpuscles. 

Enumeration of the Leucocytes. — First Method. — Enumeration 
of the leucocytes has been much facilitated by the introduction of such 
counting chambers as that of Turk, described above and pictured in 
Plate IX. If such a counter is available, a special mixing pipette, 
as I have described above, which permits of a dilution of 1 : 10, is 
not necessary. It is nevertheless convenient to have two pipettes 
of the same calibre. For counting the white cells, a dilution of 
1 : 100 is then chosen, while the red cells may be diluted to 1 : 200 
when there is reason to believe that their number is not materially 
diminished. To count the red cells in a 1 : 100 dilution, when they 
are present in approximately normal numbers, is rather taxing to 
the eyes. Such a pipette is decidedly less difficult to handle than 
one in which still lower grades of dilution can be secured. Many 
of the instruments now sent out by Leitz contain two "red" pip- 
ettes together with the Turk chamber. The ruling of this chamber 
is pictured in the accompanying plate (IX.). It will be observed 
that there are, in all, 144 large squares, the central block of 16 
of which is ruled into the smaller squares used in counting the red 

1 To focus through the thick cover Bausch & Lomb have constructed a & with a 
specially long working distance. 



144 



THE BLOOD. 



cells. The vertical and horizontal lines which serve for the ruling 
of the small squares have been extended to the margin of the 
outer ruling, but are ignored in the enumeration of the leucocytes. 
Using this chamber, the blood is diluted 100 times with Toison's 
fluid, which colors the leucocytes blue ; a drop is then mounted 
as in counting the red cells. Starting with the left upper square, 
all leucocytes are counted in the entire set of 144 large squares, 
including the 16 central squares, which are subruled into blocks 
of 16 small squares each. Leucocytes that lie in the guiding 
spaces between the large squares are ignored, and as in counting the 
red cells corpuscles on the upper and left border-lines of a square are 
counted in, while those on the right and lower lines are counted out. 
From the total number the average for 1 large square is calculated. 
As each large square represents -^-^ cbmm., the number of leucocytes in 
1 cbmm. of non-diluted blood is ascertained by multiplying the aver- 
age figure by 250 and the product by the degree of dilution — i. e. f 100. 
Example. — 50 leucocytes are seen in the 144 large squares ; the 
average for 1 would be 0.34 ; in 1 cbmm. of diluted blood there 
would be 250 X 0.34 = 85, and in 1 cbmm. of undiluted blood 
85 X 100 = 8500 leucocytes. 

Fig. 18. 




Schema of circles for counting leucocytes : a, moat surrounding central platform, b, of 
counter ; 1, starting-point. 

Second Method.— If a counting-chamber with a Turk ruling, 
or one of its modifications, is not available, but if a mechanical 
stage is at hand, the leucocytes can also be conveniently counted 
with the old pattern counter, using a red pipette for diluting the 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 145 

blood. A drop of the diluted' blood is mounted as usual. With 
the mechanical stage a field corresponding to the position of 1 in the 
accompanying diagram (Fig. 18) is then selected as the starting- 
point. The presence or absence of leucocytes is noted and the field 
changed, so that an adjoining circle is brought into view, and so on. 
In this manner at least 100 circles are gone over, using a corpuscle 
to the side or above or below as a guide to the next field. The 
total number of leucocytes is noted and the average for one circle 
calculated. If the cubic contents corresponding to each circle are 
known, the calculation of the number of leucocytes in 1 cbmm. of 
blood becomes a simple matter. The determination of the cubic 
contents corresponding to a circle is made as follows : noting the 
number of the eyepiece and the objective, the diameter of the field 
of vision is measured with a stage micrometer, or with the aid of the 
rulings of an ordinary Thoma-Zeiss counter, bearing in mind in the 
latter case that the distance between two vertical lines is -^ mm. The 
area of the circle, according to geometrical law, will then be equiva- 
lent to Tip 2 , in which tz is a constant factor — i. e., 3.1416 ; and p the 
radius, from which the corresponding cubic contents are calculated 
by multiplying the result by 0.1 — i. e., the depth of the counting- 
chamber. The resultant value, which should be ascertained for 
every instrument separately, will, of course, be constant for the sys- 
tem of lenses and the counting-chamber used. With a Bausch & 
Lomb jr (long-working distance), the 1 inch eyepiece, and 160 mm. 
tube length, the cubic contents of the field are 0.009 cbmm. 

Example. — The blood was diluted 100 times. In 100 fields 50 
leucocytes were noted — i. e., 0.5 for 1 field, or for 0.009 cbmm. ; 
in 1 cbmm. of diluted blood there would hence be 0.5 divided by 
0.009 = 55.5, and for 1 cbmm. of undiluted blood 55.5 X 100 = 
5550 leucocytes. 

Third Method. — If for any reason the 1 : 10 pipette is to be 
used in counting the leucocytes, it is necessary to choose a diluent 
which will destroy the red cells, as otherwise they would be rela- 
tively so numerous as to obscure the leucocytes altogether. For this 
purpose an 0.3 per cent, solution of acetic acid is used, which is 
tinged with gentian-violet and to which a small amount of toluol 
has been added as a preservative. W 7 ith this diluting fluid the red 
cells will be rendered invisible. The leucocytes can then be counted 
with the ordinary Thoma counting-chamber, or with that of Turk ; in 
the calculation the degree of dilution must, of course, be borne in mind. 

Cleaning of the Apparatus. — After use, the apparatus must be care- 
fully cleansed. The pipette is washed out with the diluting fluid, 
then with water, next with absolute alcohol, and finally with ether. 
The washing will be facilitated by slipping the rubber tube over the 
long arm of the pipette and blowing the contents of the bulb out of 
the short arm. In laboratories which are equipped with a suction- 

10 



146 THE BLOOD. 

pump this may be conveniently employed ; the entire process then 
occupies only two or three minutes. 

The counting-chamber is washed with water only ; alcohol and ether 
dissolve the substance with which the platform is cemented to the slide. 

Differential Enumeration of the Leucocytes. — The differential enu- 
meration of the leucocytes is usually made in dried and stained speci- 
mens. A mechanical stage is a great convenience, but not a necessity. 
The idea is to go over a large number of cells, for ordinary purposes 
not less than 500-600, to classify these, and finally to calculate the 
percentages. The cells are charted as shown below : 

S. M. (small mononuclear leucocytes) : y^ ffH ML Ml 

mi mi m mi mi =45 

L„ M. and T. F. (large mononuclear leucocytes and transition- 

forms): /%f 7/& ML == 15 

P. (polynuclear neutrophil**) : Ml 7f& Ml 7fU 7HI 

mimmimiMLMiMimiMiMi 
MirmmMimMiMimmimi 
mi m 7x1 m m mi =^ 

E. (eosinophiles) : /^ = 5 

M. (mast-cells) : // =2 

222 



: Total number of cells counted, 


222, 


, of which : 


small monos., 


45X100 

222 


= 


20.2 per cent. 


large monos., 


15 X 100 

~ 222 


= 


6.7 " " 


polys., 


155X100 

222 


= 


69.8 " " 


eosins., 


5 X 100 

222 


= 


2.2 " " 


mast., 


2X100 

222 


= 


0.9 " " 



While making a differential count it is always well to keep note 
of the time, as it is often possible in this way to form a fair idea of 
the actual number of the leucocytes without an absolute count. This, 
of course, requires a certain amount of experience in the preparation 
of the smears, which should be uniformly of nearly the same thick- 
ness. After one has then learned by control how many leucocytes 
in a blood-smear, observed within a certain length of time, may be 
considered as normal, it is not difficult to judge the grade of a hyper- 
leucocytosis by the increase in number noted within the same length 
of time. Every one must here work out his personal equation. A 
general idea of the degree of increase can, of course, be formed by 
examining the specimen with a low power — a Bausch & Lomb |-, 
for example — but in the manner indicated one gets a numerical 
expression which is at times quite helpful. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 147 

Enumeration of the Plaques. — For this purpose the method of 
Brodie and Russel has been especially advocated. The method is 
an indirect one. First, the red corpuscles are counted in the usual 
manner. A drop of the staining fluid, composed of equal parts of 
a 2 per cent, solution of common salt and a saturated solution of 
dahlia in glycerin, is then placed upon the finger, when this is 
punctured through the drop and the blood is allowed to mix with 
the reagent. In this mixture the ratio between the plaques and the 
red corpuscles is ascertained, and the total number of plaques con- 
tained in 1 cbmm. of blood determined by calculation. The plaques 
are stained the color of dahlia and can readily be counted. Rapid 
work is essential, as the staining fluid soon attacks the red corpuscles. 

Ehrlich suggests enumeration of the plaques in air-dried speci- 
mens which have been stained with acid erythrosin. Owing to the 
relatively large amount of alkali which the plaques contain, they are 
stained an intense red with this reagent (page 139). 

Rosin proposes that the air-dried specimens be fixed for twenty 
minutes by exposure to the vapors of osmic acid, and then stained 
in a concentrated aqueous solution of methylene-blue. 

These more or less complicated procedures, however, are not 
necessary as it is quite possible to count the plaques with the Thoma- 
Zeiss counter if Petrone's or Acquisto's diluent is employed (which 
see). The latter especially is useful in the modification suggested by 
Dotto, according to which 1 drop of a saturated aqueous solution 
of methyl-green is added to every 5 c.c. of the reagent. In this way 
the plaques are colored a light green. The mixing pipette should 
be washed out with the diluent before the blood is drawn up. 

The Hematocrit. 

The use of the hematocrit for counting the red blood-corpuscles 
has been repeatedly advocated, but has not met with much favor. 
The method is unfortunately inapplicable whenever there is any 
material variation in the size and form of the red corpuscles 
and whenever the number of the leucocytes is greatly increased. 
This means that the method cannot be employed in the majority 
of cases in which we are especially interested in the blood count. 
If, however, it is desired to ascertain the volume of the red cor- 
puscles in relation to the amount of plasma, the instrument will 
furnish satisfactory results. A centrifuge run by electricity is 
practically a necessity ; in this way alone is it possible to maintain 
the proper rate and uniformity of speed. Hand centrifuges are, in 
my experience, totally inadequate for the purpose, and with instru- 
ments driven by water-power it is impossible to attain a sufficient 
rate of speed. An apparatus like the one pictured in the accompany- 
ing illustration (Fig. 19) answers the purpose best. It is connected 
with the street current or with a small battery, a rheostat being 



148 



THE BLOOD. 



interposed to control the current and the rate of speed. At the 
same time a speed-indicator can be attached which strikes a bell for 
every 100 revolutions. For the hematocrit a speed of 8000 to 
10,000 revolutions per minute is required. 



Fig. 19. 



Fig. 20. 




Improved electric hsematocrit, with fender, 
rheostat, and speed-indicator. The haematocrit 
attachment replaces the urine tubes seen in 
the revolving armature. 



Daland's haematokrit. 



The hematocrit which is almost exclusively used in the United 
States is that of Daland (Figs. 20, 21, 22). It consists of a 
metallic frame which carries two glass tubes measuring 50 mm. in 
length, and 0.5 mm. in diameter. Each tube bears a scale ranging 
from to 100, the individual divisions of which are rendered more 
easily visible by a magnifying lens front. In the frame the outer end 
of each tube fits into a small depression, the bottom of which is cov- 
ered with thin rubber ; the inner ends are held in position by springs. 
The instrument is screwed to a firm table and is oiled daily when in use. 

If the patient is directly available, undiluted blood is used. The 
finger is washed with soap and water and alcohol^ as usual, and is 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 149 



freely punctured. A small rubber tube is then slipped over 
the end of one of the hematocrit tubes, which is completely filled 
by suction. The bevelled end of the tube is quickly covered with 
the finger, which has been previously lubricated with a little vaseline ; 
the rubber tube is disconnected, and the glass tube immediately fixed 
in the one compartment of the frame. Its mate is rapidly placed on 
the opposite side and the instrument rotated at a speed of from 

Fig. 21. 







Daland's hsematokrit. 



8000 to 10,000 revolutions per minute, for three minutes, when the 
volume is directly read off. In normal individuals the volume of 
the red corpuscles is approximately 50 per cent., so that in a given 
case a proportionate expression of the percentage of corpuscles, as 
compared with the normal, can be obtained by multiplying the figure 

on the scale by 2. 

Fig. 22. 




Daland's hsematokrit. 

If the patient is not directly available, the blood is diluted with 
an equal volume of a 2.5 per cent, solution of potassium bichro- 
mate, as proposed by Daland. As Ewing suggests, this can be 
done with the pipette which accompanies the Thoma-Zeiss blood- 
counter. In the case of the red pipette the capillary tube is filled 
with blood to the mark 1, then a small air-bubble is drawn in, 
followed by another tube-length of blood. Three or four volumes 
of blood are obtained in this way and diluted at once with an equal 
quantity of the bichromate solution. In the case of the white 
pipette a single tube-length of blood and the diluent is sufficient. 
Blood and diluent are thoroughly mixed, care being had not to 
include any air-bubbles. In this form the blood is carried to the 
laboratory, where both tubes are filled by allowing the drops to flow 
in from the point of the pipette. To obtain the percentage volume, 
the resultant figure is in this case, of course, multiplied by 4. 

In the case of normal blood it has been ascertained that 1 per 
cent, by volume, as read off from the scale, corresponds to almost 



150 THE BLOOD. 

100,000 red corpuscles per cbnim. ; to obtain the total number of 
red cells per cbmni., it is hence only necessary to add five ciphers to 
the percentage indicated on the scale. 

Example. — Undiluted blood was used ; the reading on the scale 
was 45. The volume per cent, of the red corpuscles would hence 
be 90, and the number of red cells per cbmm. 4,500,000. 

But, as I have pointed out, this calculation presupposes that the 
size and form of the red cells are practically normal, and that the 
leucocytes are not materially increased. 

With normal blood the leucocytes appear only as a narrow, indis- 
tinct milky band at the central end of the column of red cells, which 
with a material increase of the leucocytes becomes more marked and 
reaches its greatest extent in well-marked cases of leukaemia. 

Aspelin has recently suggested that with a suitable modification 
of the Daland apparatus quite accurate leucocyte counts can be ob- 
tained by centrifugation ; but bearing in mind the variations in the 
size of the different leucocytes and the varying degree in which the 
different forms take part in the production of the different types of 
hyperleucocytosis, it is evident at once that still less is to be antici- 
pated from the centrifugal method in this direction than in the case 
of the red cells. 

Literature. — Hedin, Arch. f. ges. Phys., vol. xl. p. 360. Gartner, Wien. klin. 
Woch., 1892, No. 2. Daland, Fort. d. Med., 1891, No. 21. Aspelin, Zeit. f. klin. Med., 
1903, vol. xlix. p. 393. 

Estimation of Haemoglobin. 

Hsemoglobinometers. — While it is usually possible to form a 
fairly clear idea of the degree of anaemia by direct inspection of the 
patient, the appearance of the mucous surfaces, etc., it is often de- 
sirable to obtain more definite information, and, above all, a numer- 
ical expression of the extent of the anaemia. This is especially 
important in the diagnosis of certain forms of anaemia, in which the 
" color index " plays an important part — i. e., the ratio between the 
percentage of haemoglobin and the percentage of the red corpuscles 
as compared with the normal. To this end, special instruments have 
been devised, which are termed hcemoglobinometers or hcemometers. 
Of the various forms which are now in the market, the haemoglobin- 
ometer of Dare is probably the best, and is rapidly replacing the old 
instrument of v. Fleischl, which for many years was the standard. 
It is more exact and more convenient. Miescher's modification of 
the Fleischl instrument is possibly still more accurate, but too 
costly for general adoption. The little instrument of Gowers, 
which has long been in the market, when obtained from a reliable 
source will also furnish good results, and is warmly commended 
by Sahli, Emerson, and others. Unfortunately many of those which 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 



151 



Fig. 23. 



have been placed on sale are worthless. Oliver's instrument has 
some advantages over the Fleischl, but none over the Dare. The 
Talquist method is warmly commended by Cabot, and may be used, 
to advantage in routine work by the general practitioner ; for exact 
work it is insufficient. 

Dare's Hsemoglobinometer. — The essential parts of Dare's haemo- 
globinometer (Fig. 23) are an automatic pipette for collecting the 
blood (Fig. 24) and a graduated color scale (Fig. 25) to measure 

the corresponding percentage 
of haemoglobin. This latter 
reads from 10 to 120, the 100 
mark corresponding to the 
color of a solution of 13.77 
grammes of haemoglobin in 
100 c.c. of serum. The various 
shades of color corresponding 
z to the scale are obtained by 
rotation of a prismatic glass 
semicircle tinted with golden 
purple of Cassius (Fig. 25, 
E), which is secured to a thin 
white glass disk (I). The 
numerical scale is placed on 
the edge of a corresponding 
semicircle (H) of thick white 
glass (F). This part of the apparatus is enclosed in a dust-proof 
hard-rubber case, and is rotated from the outside by the aid of 
a rubber-covered roller which runs on the edge of the disk and is 
turned by a milled wheel at R (Fig. 23). In the rubber case 
is a little circular window through which the color of the prism 




Dare's hsemoglobinometer. 




Fig. 25. 




Automatic pipette. 



Graduated color scale. 



is viewed by means of a small telescoping camera tube (Fig. 26, N), 
provided with a magnifying lens of low power. The color aperture 
represents a surface about equal to 3 per cent, of the color scale. 
Looking through the tube a corresponding window will be seen side 
by side with the one through which the color scale is visible. In front 
of this the blood pipette is secured. The essential part of this is an 



152 



THE BLOOD. 



oblong plate of white glass (Fig. 24, A), into the end of which a 
depressed surface of measured depth is ground, the floor being ex- 
actly parallel to the plane surface of the glass. This depression 
forms a capillary chamber (D) when the transparent glass plate (B) 

is firmly clamped upon it by the pipette 
clamp C ; it is filled by capillary attrac- 
tion when either of the three free edges 
is touched to the blood drop. The 
pipette is held in position on the stage 
of the instrument by guides which run in 
grooves on the lower part of the clamp. 
The plate of white glass is toward the 
light. 



Fig. 27. 




% 




pssss. 




M' 


M'/ 








N 




c 






1 



Horizontal section of Dare's 
hsemoglobinometer ( on a level with 
centre of comparison apertures) : 
J, candle; K, white glass disk of 
color prism; L, color prism; M, 
aperture through which color of 
blood film is viewed ; M', aperture 
through which the illuminated 
color prism is viewed ; N, camera 
tube ; 0, transparent glass of 
pipette ; P, white glass of pipette. 




Filling the automatic blood pipette. 



The camera tube screws into a movable shutter (Fig. 23) ; when 
this is swung outward, the two apertures become visible through 
which the blood and the colored scale are viewed. 

In front of the pipette a candle is clamped in such a position that 
both the blood and the color scale are equally illuminated. 

Method of Use. — As the comparison of the color of the blood 
with that of the color scale should be made as soon after filling the 
pipette as possible, the apparatus is prepared for use beforehand by 
screwing the camera tube into place and adjusting the candle ; this 
should be at such a level that the blue flame of the candle is below 
the color aperture, care being taken to have the wick of proper length 
(half inch) and not charred at the tip. Curved or eccentric wicks 
should be turned so that the intensity of light in a vertical position 
is midway between the two color apertures. 

The glass plates of the pipette having been thoroughly polished and 
refastened in the clamp, the finger or ear is freely punctured as usual 
and the capillary space of the pipette filled with the blood, by hold- 
ing one of the three edges horizontally to the drop (Fig. 27). Any 
blood adhering to the flat surfaces of the glass plates is carefully 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 153 

wiped away and the pipette placed in position. The candle is 
lighted, the shutter thrown out, the camera tube focussed, and the 
color of the blood (on the left) compared with the color scale (on the 
right). The two are matched by rotating the color disk by means 
of the milled wheel, which should be done in an abrupt manner, 
and frequently resting the eye. To this end, the shutter is dropped 
and thrown out again as the case may be. The examination need 
not be conducted in a darkened room, but it is important to turn the 
instrument toward a dark background, so as to eliminate all direct 
or reflected light. The reading is indicated by the bevelled edge of 
the rectangular opening on the side of the case ; the figure immedi- 
ately beneath this represents the percentage of haemoglobin. Im- 
mediately after use, the two glass plates of the pipette are cleansed 
with water and a little acid alcohol, dried, and again replaced. Fur- 
ther details in regard to technique accompany the instrument. 

The amount of haemoglobin in grammes corresponding to the 
readings is calculated according to the equation, 100 : 13.77 :: p :x, 
and x = 0.137 p, where p represents the reading actually noted and 
x the corresponding amount of haemoglobin in 100 grammes of 
blood. 

My personal experience with the instrument has been quite satis- 
factory. The readings are somewhat higher than those obtained 
with the Fleischl instrument and represent the actual condition more 
exactly. 

Literature. — A. Dare, Phila. Med. Jour., Sept. 22, 1900. 

Fleischl' s Hsemoglobinometer. — The principle underlying the 
v. Fleischl method is essentially the same as that of the Dare 
method ; the color of the blood is compared with the color of a glass 
wedge stained with the golden purple of Cassius or a similar pig- 
ment, a scale indicating the corresponding amount of haemoglobin. 
With the Fleischl method, however, diluted blood is used, which is 
one of the disadvantages of the method. 

The instrument (Fig. 28) consists of the glass wedge a, to which 
a scale, 6, is attached, ranging from to 120, being placed at the 
thinnest, 120 at the thickest portion of the wedge. By means of a 
rack and pinion this may be made to slide from side to side beneath 
a platform corresponding to the stage of a microscope. In the 
centre of the platform there is a circular opening into which artifi- 
cial light (daylight is not permissible) is projected from a circular 
plate of plaster-of-Paris mounted beneath, in the position of the 
mirror of the microscope. Into the circular opening a metallic tube, 
1.5 cm. in height, is fixed, which is closed at the bottom with a 
plate of glass and divided into two equal compartments by a metal 
partition. One compartment receives the light through the glass 



154 



THE BLOOD. 



wedge— the red chamber ; the other, direetly from the plaster-of- 
Paris reflector — the white chamber. 

Capillary pipettes of known capacity accompany the instrument. 
This capacity is somewhat variable and is indicated on the handle 
of each, which number must correspond with that marked on the top 
screw-head of the instrument. Generally speaking, the capacity of 
each pipette is such that with the blood of a perfectly normal indi- 
vidual the mixture of blood and water in the white chamber will 
correspond in color to that of the colored wedge at the mark 100 
(a 13.77 per cent, solution of haemoglobin). 

Fig. 28. 




v. Fleischl's hsemometer. 



The pipette is filled by capillary attraction from a drop of blood 
obtained in the usual manner. If on trial it is found that the blood 
does not immediately run up in the tube, this is repeatedly washed 
out with water and then dried. If this is always done after the 
examination, the pipette will be in working order on the next 
occasion. While filling the pipette care should be had that it is not 
immersed in the blood, but only brought in contact with it. The 
two compartments of the cell having been previously partly filled with 
water, the charged pipette is at once placed in the white chamber 
and rapidly moved to and fro until the blood is well mixed with 
the water. Any trace remaining in the pipette is carefully washed 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 155 

out with water by the aid of a medicine-dropper. The contents of 
the chamber are stirred with the handle of the pipette when both 
compartments are filled with water, using the same dropper, so that 
there is a convex meniscus over each. The color of the blood is 
then matched on the Avedge, which should be moved by quick turns 
of the adjustment-screw rather than in a gradual way, as the eye will 
otherwise be less apt to appreciate fine shades of difference. Day- 
light, as I have said, is not permissible ; a candle- or gas-flame of 
moderate intensity placed about a foot and a half distant is best. 
The eye should be perpendicularly above the cell, and it is well to 
view the colors through a paper tube which is placed over the two 
compartments. The number facing the notch in the little well 
immediately behind the cell indicates the percentage of haemoglobin. 
The readings corresponding to the middle portion of the wedge are 
apt to be more nearly correct than the lower values. For this 
reason it is well, when a preliminary examination has shown a low 
figure, to repeat the test, using two or three pipettefuls of blood 
instead of one, the result, of course, being divided by 2 or 3, as the 
case may be. On the whole, the Fleischl method furnishes results 
which are somewhat lower than those obtained with the Dare ; this 
is true especially of the older models, with which a percentage of 
100 was only rarely observed. The instruments of more recent 
construction, however, are much better. Personally I regret to see 
the Fleischl apparatus supplanted by newer instruments ; it was con- 
venient and neat. It has its defects, to be sure, and it is unfor- 
tunate that the Miescher modification, in which these have been elim- 
inated, and which unquestionably gives the most accurate, results, is 
still so costly that its general use is out of the question. 

Oliver's Hsemoglobinometer. — With this method the color of the 
blood in definite dilution is compared with a series of tinted glass 
standards. The principal advantage of the method lies in the fact 
that the color of the blood is compared at one time with one single 
tint. Two sets of standards are furnished ; one intended for day- 
light, the other for candle-light, which latter should be chosen by 
preference as it yields more accurate results. Each set consists of 
twelve shades of color, the glass disks being mounted in two sets on 
a plaster-of-Paris background. They represent the color of a solution 
of haemoglobin with a percentage running by tens from 10 to 120 
(Fig. 29). Values intermediate between any two tens are obtained 
by superimposing riders of colored glass upon the disks, which 
represent 2.5 and 5 per cent, of haemoglobin in the upper half of 
the scale, but twice this amount in the lower half. 

A diluting chamber for diluting the blood, a pipette of 5 cbmm. 
capacity, a dropper, and a lancet accompany the color scale. The 
diluting chamber has a white background of plaster-of-Paris and a 
cover of blue glass. The procedure is the following : The finger is 



156 



THE BLOOD. 



punctured as usual and the pipette filled by capillary attraction, 
taking care that it only touches and is not immersed in the drop. 
The dropper, filled with water, is immediately attached to the narrow 



Fig. 29. 




Oliver's hfemoglobinometer : a, set of standard colored disks ; b, lancet ; c, capillary pipette ; 
d, dropper ; e, mixing chamber. (Ewmg.) 

end of the pipette, and the blood mixed with water forced drop by 
drop into the diluting chamber ; care must be had that the blood is 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 157 

not drawn into the pipette by adjusting the rubber end-piece while 
holding the pipette by its glass tube. The chamber is filled with 
water, the solution stirred with the handle of the pipette, and the 
blue-glass cover adjusted. When properly prepared, a tiny bubble 
of air should be included, showing that the chamber has not been 
overfilled. The candle is now placed at a convenient distance and 
the color of the diluted blood compared with that of the standard 
disks, looking through a collapsible camera tube. If it matches the 
color of any one of the disks, the percentage is read oif at once. If, 
however, it is lighter than 40, for example, but darker than 30, one 
of the riders is placed upon 30, and a corresponding plate of un- 
stained glass upon the diluting chamber, to compensate for the thick- 
ness of the rider. If the color cannot be matched in this manner, 
the average between the paler shade and the darker shade above is 
taken, remembering that on the lower half of the scale the actual 
value of the rider is double its face value. The room need only be 
darkened in part. 

The method is less convenient than that of Dare or Fleischl, but 
yields good results ; variations due to unavoidable error do not 
amount to more than 2 per cent. The cost of the instrument, how- 
ever, is too great for its general adoption. 

Gowers' Haemoglobinometer. — The apparatus (Fig. 30) consists of 
two glass tubes (A and B) which are of the same diameter. One of 
these (A) is closed and contains a solution of picrocarmine-glycerin, 
the color of which corresponds exactly to that of a 1 per cent, solu- 
tion of normal blood. The other tube is provided with an ascending 
scale of 120 divisions, each degree corresponding to 20 cbmm. A 
capillary pipette marked at 20 cbmm., a guarded lancet, a dropping- 
bottle, and a small stand accompany the instrument. On the stand 
the tubes can be so adjusted that it is possible to view them at an 
angle where the adjoining sides appear to overlap. 

The finger is punctured as usual and the pipette filled to the 20 
cbmm. mark ; the blood is immediately discharged into the gradu- 
ated tube and mixed with a few drops of water that has been pre- 
viously placed there. The pipette is carefully washed out with 
water and the washings added to the blood ; this is then further 
diluted with water drop by drop until the color of the blood, when 
held to the light or against a sheet of white paper, exactly matches 
that of the standard solution. After the addition of each drop the 
tube is inverted several times, closing the open end with the thumb ; 
any adhering drops are wiped off against the edge of the tube so 
that they will flow back. The division on the scale ultimately 
reached indicates the percentage of haemoglobin. 

With very low percentages it is advised to use double the amount 
of blood, when the final result is, of course, divided correspondingly. 

The method, as I have indicated above, is satisfactory if the 



158 



THE BLOOD. 



instrument has been obtained from a reliable source. Its low cost 
makes it especially serviceable in large clinics and for purposes of 
teaching in the clinical laboratory. 

Talquist's Method. — The principle of the method is essentially the 
same as that underlying the Oliver method. The color of the blood, 
in this case undiluted, is compared with a series of lithographed 
standard tints, which represent a scale ranging by tens from 10 to 
100. The technique is very simple : drops ot blood are received on 
pieces of white filter-paper of suitable thickness which accompany 
the color scale, and are compared with the tints on the plate, using 
ordinary daylight. 

Fig. 30. 




Gower's hsemoglobinometer. 



so 



Accuracy is, of course, not to be expected from so crude a method, 
that its use is of necessity limited. It will suffice in a very 
general way to control the result of treatment, but it is inapplicable 
in the determination of the color index. 

Estimation of Blood-iron with Jolles' Ferrometer. — The estimation 
of the haemoglobin from the amount of blood-iron, as originally sug- 
gested by Jolles, is unfortunately not possible, as it has been shown 
that constant relations between the two bodies do not exist. All the 
iron of the blood is not present in this form, nor does it all occur 
in the form of colored compounds. Jolles' method of estimating 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 



159 



the total amount of blood-iron deserves consideration, however, as 
it is a practical method and discloses facts which are of clinical in- 
terest. It is desirable that it should be introduced in the clinical 
laboratory as a routine method. 

The principle is the following : a small amount of blood is 
incinerated, and the remaining red oxide of iron brought into 



Fig. 31. 




Jolles' ferrometer. 



solution with a little monacid potassium sulphate. In this solu- 
tion the iron is then estimated colorimetrically by means of a spe- 
cial apparatus — the ferrometer. As will be seen from the accom- 
panying illustration (Fig. 31), this consists of two glass tubes, C 
and (7, which are of the same diameter throughout, and closed at 
the bottom with small glass plates, held in position by means of 
screws, as in the polarimetric tubes. Tube C is of 15 c.c. 
capacity, while tube C is a little longer and holds about 16 c.c. 
Both are graduated in 0.5 c.c. Tube C is provided with an over- 
flow tube near the bottom, which carries a stopcock. Both are fitted 
into the perforated metallic plate D, and are surrounded by a cas- 
ing, so as to exclude light from the sides. Below the plate is a 
plaster-of- Paris reflector, which can be turned with the screws K 
and K f . Tube C receives the iron solution, obtained from the 
blood, and is closed with an accurately fitting glass disk, while in 
O is placed the iron solution used for comparison. This is allowed 



160 THE BLOOD. 

to flow away through the overflow tube (iJ) drop by drop until the 
color in the two tubes is the same. But as the color in (7, owing 
to the meniscus which is formed, would be less sharply defined than 
in C y the tube O is furnished with a cylindrical float of aluminum, 
which is closed above and below with glass disks. This float dis- 
lodges about 1 c.c. of fluid, and it is for this reason that tube O is 
a little longer than tube C. 

A capillary pipette and the necessary additional apparatus, as well 
as reagents, accompany the instrument, which is made by Reichert, 
of Vienna. 

Method. — In order to procure the necessary amount of blood, 
viz., 0.05 c.c, which is obtained by simple puncture of a finger or the 
ear, Jolles recommends that the capillary tube be first filled beyond 
the mark, and to close the pinchcock on the rubber tube at once. 
The excess of blood is then allowed to flow from the tube, and the 
tip is carefully wiped with filter-paper. The 0.05 c.c. is placed in 
a platinum crucible, any traces that may remain adherent to the 
tube being washed out with a little distilled water. 1 

The blood is evaporated to dryness over a plate of asbestos, 
at first with a small flame. The crucible is placed on a pipe-stem 
triangle, and the residue carefully incinerated. One of the accom- 
panying powders, containing 0.1 gramme of monacid potassium 
sulphate is now added. The mixture is cautiously heated with 
a small flame until the powder begins to liquefy, when stronger 
heat is applied and the mass congeals. This step is completed in 
one or two minutes. On cooling, the material is washed into the 
cylinder (7, through a small funnel with the aid of a little hot dis- 
tilled water, and diluted to the mark 10. The tube O is charged 
with 1 c.c. of the comparison-solution, and likewise filled to the 
mark 10 with hot distilled water. This solution contains 0.0005 
gramme of iron and 0.1 gramme of monacid potassium sulphate, in 
every cubic centimeter. 

To each cylinder are then added 1 c.c. of hydrochloric acid (1:3) 
and 4 c.c. of a solution of ammonium sulphocyanide (7.5 grammes pro 
liter). The tube C is now closed with the glass disk, care being 
taken to exclude bubbles of air, when the mixture is thoroughly 
shaken and the tube fixed in the metallic plate. Tube C is likewise 
closed with a glass disk ; its contents are well agitated, the disk is 
removed and replaced by the carefully dried float. This should be 
placed upon the fluid slowly and with a screwing motion, so as to 
exclude bubbles of air. After this tube has also been placed in 
position the reflector is adjusted, and so much of the comparison- 
solution allowed to escape as to make the color in the two tubes the 

1 The pipette should always be eleansed immediately after use. It is best washed 
out with dilute sulphuric acid (10 per cent.), then with dilute sodium hydrate solution 
(5 per cent.), and finally with alcohol and ether. 



MICROSCOPICAL EXAMINATION OF THE BLOOD. 161 

same. O is then removed from its base and the reading taken. In 
the table below, the corresponding amount of iron in 1000 c.c. of 
blood may be directly read off. Should it be desired to obtain the 
percentage by weight, the specific gravity of the blood should first be 
ascertained, and the necessary calculation made according to the 

equation D : V: : 100 : x, and x= '- — , in which JD represents 

the specific gravity, and V the percentage by volume. The resulting 

differences, however, are so small that they may be neglected, and 

for practical purposes it will be sufficient to assume a specific gravity 

of 1.050, and to read off the percentage by weight directly. To 

this end, the second column in the table has been constructed. 

Table to ascertain the Amount of Ikon in 1000 c.c. of Beood, and the 
Percentage by Weight, from the Number of c.c. of the Comparison- 
solution used. 



C.c. of comparison- 


Iron in 1000 c.c. 


Iron-percentage 


solution used. 


of blood. 


by weight. 


15.0 


1.000 


0.0952 


14.5 


0.967 


0.0920 


14.0 


0.933 


0.0889 


13.5 


0.900 


0.0857 


13.0 


0.867 


0.0825 


12.5 


0.833 


0.0794 


12.0 


0.800 


0.0762 


11.5 


0.767 


0.0730 


11.0 


0.733 


0.0698 


10.5 


0.700 


0.0666 


10.0 


0.667 


0.0635 


9.5 


0.633 


0.0603 


9.0 


0.600 


0.0571 


8.5 


0.567 


0.0540 


8.0 


0.533 


0.0508 


7.5 


0.500 


0.0475 


7.0 


0.467 


0.0444 


6.5 


0.433 


0.0412 


6.0 


0.400 


0.0381 


5.5 


0.366 


0.0349 


5.0 


0.333 


0.0317 


4.5 


0.300 


0.0285 


4.0 


0.266 


0.0254 


3.5 


0.233 


0.0222 


3.0 


0.200 


0.0191 


2.5 


0.166 


0.0158 


2.0 


0.133 


0.0127 


1.5 


0.100 


0.0095 


1.0 


0.067 


0.0063 



More recently Jolles has modified his ferrometer in such a manner 
that the comparison of the sulphocyanide solution, obtained from the 
blood, is made with the colored wedge of FleischPs hsemometer. The 
new instrument (Fig. 32) he terms the clinical ferrometer, and, as 
made by Reichert in Vienna, it can readily be transformed into the 
hsemometer proper. Full directions accompany the apparatus. The 
results are expressed in relative terms, the number 100 on the scale 
U 



162 



THE BLOOD. 



corresponding to 0.0425 per cent, by weight of iron. Some of the 
results which have been obtained with the clinical ferrometer are 



Fig. 32. 




given below, together with the corresponding figures indicating the 
amount of haemoglobin. 

Ferrometer Hsemometer 

number. number. 

Normal 103.0 100 

Normal 92.6 105 

Normal 95.5 100 

Normal 110.0 105 

Normal 83.8 92 

Chlorosis 32.1-68.2 30-65 

Simple anaemia 33.2-74.7 15-40 

Icterus 55.0 80 

Leukaemia 40.7 32 

Leukaemia 38.6 35 

Pseudoleukaemia 77.24 75-80 

Severe diabetes 78.7 30 

Severe diabetes 91.4 35-40 

Parenchymatous nephritis 51.7 50 

These figures at once illustrate the lack of relationship which exists 
between the amount of haemoglobin and that of the blood-iron as a 
whole, 



3IICR0SC0PICAL EXAMINATION OF THE BLOOD. 163 

Jellineck has made a careful comparative study of the blood with 
Jolles' instrument and v. FleischFs haemometer and arrived at t ome 
very interesting conclusions. In diabetes he thus found that the 
amount of iron steadily diminishes, although the hsemoglobinometer 
gives higher readings. In a case of malaria the iron remained con- 
stant before and after the chill, while with v. Fleischl\s instrument 
variable results were obtained. In two cases of leucocytosis the 
ferrometer gave low readings, and in eight cases of secondary anaemia 
the hsemometer gave much higher values than the ferrometer. 

In a series of cases Jolles also examined into the presence of iron 
in the serum, by centrifugating a given volume of blood mixed with 
an 0.8 per cent, salt solution, and found that in health the serum 
contains no iron. In three cases of chlorosis, in one case of leukae- 
mia, in one of neoplasm, and one of interstitial nephritis, negative 
results were likewise reached. In two cases of severe diabetes, on 
the other hand, notable quantities were found. 

Literature. — A. Jolles, "Ferrometer," Deutsch. med. Woch., 1897, No. 10; Ibid., 
1898, No. 7. Hladik, " Untersuchungen iiber d. Eisengehalt d. Blutes gesunder Men- 
schen," Wien. klin. Woch., 1898, No. 4. S. Jellineck, " Ueber Farbekraft und Eisen- 
gehalt d. Blutes," Ibid., Nos. 33, 34. A. Jolles, " Vereinfachtes klin. Ferrometer," 
Berlin, klin. Woch., 1899, No. 44, p. 965. 

Kryoscopic Examination of the Blood. 

The kryoscopic examination of the blood has for its object the 
determination of the molecular concentration, and hence of the 
osmotic pressure of the blood. The method is essentially based 
upon the observations of Raoult : (a) that all solid, liquid, or gase- 
ous substances when dissolved in a liquid will lower the freezing- 
point of that liquid ; (6) that the degree to which the freezing-point 
is lowered is dependent upon the amount of substance which is 
present in solution ; and (c) that equimolecular solutions have like 
freezing-points . l 

It follows that the freezing-point of a solution furnishes an index 
of its molecular concentration, and hence also of its osmotic press- 
ure, as this has been shown by van't Holf to be proportionate to 
the number of molecules present. 

The degree to which the freezing-point is lowered is designated 
by the letter /\. In the case of normal blood this varies between 
— 0.56 and — 0.58° C, as compared with distilled water. A further 
depression is probably always indicative of renal insufficiency ; it is 
a symptom of decided value and deserves more general considera- 

1 Solutions are termed equimolecular when for a constant quantity of the solvent 
they contain such quantities of substance in solution that these bear the same ratio 
to each other as their molecular weights. Example : The molecular weight of sodium 
chloride is 58.5 and of sodium carbonate 106 ; if we dissolve these quantities or the 
same multiples of each in a constant quantity of water, such solutions would be equi- 
molecular. 



164 



THE BLOOD. 



Fig. 33. 



tioa. In the domain of renal surgery especially the study of kryos- 
copy of the blood is especially important. Of foreign investigators, 
Kummel more particularly has pointed out the value of the method 
in this field. As the result of 265 freezing-point determinations 
of the blood, in 170 cases in which various operations were per- 
formed upon the kidney and in which a direct examination of the 
organ was possible, he concludes that kryoscopy furnishes the most 
important index of renal insufficiency as compared with all other 
modern methods. Other observers, such as Casper and Bichter, 
Tinker, and others, have arrived at similar conclusions. To Koranyi, 
however, belongs the credit for the introduction of kryoscopy into 
the clinical laboratory and its application to the study of renal dis- 
eases. Senator, Claude and Balthazar, Albarran, Kovesi, Linde- 
manu, Waldvogel, and others have materially contributed to estab- 
lish its value as a clinical method. 

Method. — In the clinical laboratory a modification of Beck- 
mann's apparatus is most conveniently employed (Fig. 33). Its 

essential parts are : (a) a Heidenhain 
thermometer graduated in hundredths and 
reading from — 1° to — 5° C. ; (b) a plati- 
num wire loop for stirring ; (c) a test-tube 
which is closed by a stopper through which 
the thermometer and stirring-wire pass, 
and which in turn is placed in a second 
larger tube (d) so as to be surrounded 
by an air-space. The jar / is filled with 
a freezing-mixture of salt and ice, the 
temperature of which should lie between 
— 2° and — -5° C. Into this is placed 
the second tube d. The test-tube c is 
charged with 20 c.c. of blood (if only 
10 c.c. are available, this amount may 
suffice), obtained by means of a large 
aspirating-syringe from one of the veins 
j|\ I pj | near the bend of the elbow, as in the case 

of a bacteriological examination of the 
blood (page 166) ; the thermometer is in- 
troduced and the stirring- wire adjusted. 
The tube is placed directly in the freezing- 
mixture until the mercury leaves the reser- 
voir bulb ; this is done to save time. It is 
then adjusted in the second tube d, as shown in the illustration, and 
the blood constantly stirred with the platinum wire. The temperature 
falls more or less rapidly below the freezing-point before actual 
freezing takes place ; as this occurs it suddenly rises again owing 
to liberation of heat, and then remains constant for some time. This 




'"*''5£:,'"' — ■ 
Beckmann's apparatus. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 165 

point represents the true freezing-point. Later, if the tube is 
allowed to remain in the freezing-mixture, the temperature may fall 
to that of the latter. The difference between the freezing-point of 
distilled water and that of the blood is /\ . 

In every case it is necessary to determine the true zero for each 
instrument separately, as this often varies somewhat owing to un- 
avoidable errors incident to its construction. To this end, the tube 
c is charged with three to four times the amount of distilled water 
which is necessary for one examination. The greater portion of this 
is frozen ; the liquid portion is thrown away ; the frozen water 
is allowed to thaw and is again frozen in part, a portion being again 
thrown away ; the remainder is sufficiently pure for the examina- 
tion. 

The freezing-mixture is prepared by packing alternate layers 
of ice and salt into the jar /around the tube d, which is held in 
position while the ice is packed. Ice and salt are finally thoroughly 
mixed by stirring with a heavy wire ring and rod (g). If several ex- 
aminations are to be made, the water which separates out is poured 
off and replaced by an additional amount of salt and ice. 

The method is quite expeditious, and if everything is previously 
prepared, the examination does not occupy more than ten or fifteen 
minutes. 

Literature. — v. Koranyi, Zeit. f. klin. Med., 1897, vol. xxxiii., and 1898, vol. 
xxxiv. Lindemanu, Deutsch. Arch. f. klin. Med., 1899, vol. lxv. Albarron, Annal. 
d. mal. genito-urin., 1899. Senator, Deutsch. med. Woch., 1900, vol. xxvi. p. 48. 
Claude and Balthazar, Presse med., 1900, vol. xviii. p. 85. Casper and Richter, Funk- 
tionelle Nierendiagnostik, Berlin u. Wien, 1901. Kiimmel, Centralbl. f. Chir., 1902, 
vol. xxix. p. 121 of Beilage. Tinker, Johns Hopkins Hosp. Bull., 1903, vol. xiv. 
p. 162. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 

Typhoid Fever. 

Recent researches have shown that in typhoid fever the specific 
organism (Plate XV., Fig. 3) can be isolated from the blood di- 
rectly in a fairly large percentage of cases and at a time when the 
Widal reaction (see below) may not as yet be obtainable, viz., on the 
fifth day of the disease. Schottmuller thus found the organism in 
40 of 50 cases, Castellani in 12 of 14, Kuhnau in 11 of 41, Cour- 
mont in all of 9 cases, Auerbach and Unger in 7 of 10, and Cole in 
11 of 15 cases. Neuhaus, Neufeld, Curschmann, Rumpf, and others 
had previously shown that the bacillus may at times be cultivated 
from the blood taken from the roseolar spots. More recently Po- 
lacco and Gemelli report that with a modification of Neufeld's 
method they obtained the bacillus from the rose spots in every one 
of 50 typhoid patients. 



166 THE BLOOD. 

The blood is withdrawn by means of a sterilized syringe from one 
of the superficial veins of the arm ; 300 to 500 c.c. of bouillon are 
inoculated with from 2 to 4 c.c. of the fresh blood and examined after 
from eighteen to twenty-four hours. If a negative result is obtained 
in the hanging drop, a further examination is made twenty-four 
hours later. At first the bacilli are but little active, but on further 
cultivation and reinoculation their motility increases. For purposes 
of identification they are grown on agar slant, in milk, bouillon, 
glucose, and further tested with an actively agglutinating serum 
(see below). It is interesting to note, however, that their tendency 
to agglutination is almost invariably much inferior to that of bacilli 
which have been maintained for a long time on artificial media. 
Courmont thus notes that they were commonly agglutinated with a 
dilution of 1 : 50 by a serum which agglutinated laboratory bacilli 
at 1 : 200. 

Cole uses from 8 to 10 c.c. of blood, which is immediately diluted 
with bouillon contained in Erlenmeyer flasks, about 150 c.c. of bouil- 
lon being used for each flask. From one to six flasks are prepared, 
the dilution being 1 : 75 to 1 : 150. The flasks are then well skaken 
and placed in an incubator for twenty-four hours, after which, if the 
bouillon is cloudy, agar plates are made. With this technique a 
positive result can be obtained in thirty-six hours. 

Literature. — Neuhaus, Berlin, klin. Woch., 1886, Nos. 6 and 24. Schottmiiller, 
Deutsch. med. Woch., 1900, No. 32. Castellani, cited in Presse med., June, 1900. 
Auerbach u. Unger, Deutsch. med. Woch., 1900, No. 29. Cole, Johns Hopkins Hosp. 
Bull., 1901, p. 203. Courmont, Jour. d. physiol. et d. pathol. gen., 1902, vol. iv. p. 
155. Polacco and Gemelli, Centralbl. f. inn. Med., 1902, vol. xxiii. p. 121. 

Widal's Serum Test. — Of greater practical utility than the culti- 
vation of the typhoid bacillus from the blood is the fact that the 
blood-serum of patients affected with typhoid fever possesses the 
property of causing arrest of motility and agglutination of the spe- 
cific bacilli. This observation, originally made by Pfeiffer, was first 
utilized for diagnostic purposes by Widal, in 1896. The method 
which bears his name has now been quite generally adopted in the 
clinical laboratory, and must be regarded as a most valuable aid in 
the diagnosis of typhoid fever. The reaction occurs in over 95 per 
cent, of undoubted cases, and may appear as early as the first day 
of the disease, meaning thereby the first day that the patient spends 
in bed or the fifth day of general malaise. Such instances, however, 
are very uncommon, and, as a general rule, a positive result is ob- 
tained only after the fifth or sixth day in bed. In a small number of 
positive cases, on the other hand, the patient may pass through the 
entire course of the disease, and present typical clumping only dur- 
ing convalescence or a subsequent relapse. In every case, therefore, 
in which no reaction is obtained upon first trial, the test should be 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 167 

repeated at regular intervals throughout the disease until a definite 
result is obtained. Intermittence of the reaction, moreover, is very 
common, and emphasizes still further the necessity of frequent exami- 
nations in apparently negative cases. 

While in some instances the reaction disappears very soon after 
the temperature reaches normal, and even earlier, it generally con- 
tinues into convalescence, and may be observed for months and years 
after the attack. Cases have thus been recorded in which a positive 
reaction could be obtained as long as thirty-seven years after in- 
fection. 

The question, whether or not WidaFs reaction is a specific reac- 
tion of the typhoid organism, can, I think, be answered in the 
affirmative, notwithstanding the facts that at times cases of true 
typhoid fever are seen in which no clumping is obtained, and that the 
reaction has been observed in cases which were apparently non- 
typhoid. Such exceptions, no doubt, are due in part to faulty tech- 
nique, viz., to too low a degree of dilution of the serum, the use of 
old or impure cultures, too long a time-limit of observation, single 
negative tests, etc. On the other hand, there can be no doubt that 
typhoid bacilli are at times present in the body without giving rise 
to symptoms of typhoid fever. In a case of cholelithiasis, reported 
by Cashing, typhoid bacilli were thus found in the gall-bladder, and 
distinct clumping was observed with a dilution of 1 : 30, although 
no history of typhoid fever could be obtained. There can further 
be no doubt that individuals exist who are naturally immune against 
typhoid fever, and that some of the positive results which have been 
obtained in perfectly healthy individuals who have never had typhoid 
fever may be explained in this manner. 

While the reaction may hence be regarded as a specific infectious 
reaction of the typhoid organism, nevertheless its value in diagnosis 
is limited. This is owing largely to the fact that in many cases a 
positive result is not obtained before the end of the second or third 
week, and may even be delayed until a relapse occurs. Its per- 
sistence for years after infection is also an obstacle to its general 
utility, not to speak of its occurrence in apparently healthy individ- 
uals and in diseases in which an association with the typhoid organ- 
ism is not apparent. A rather interesting apparent exception to the 
rule that the Widal reaction is only obtained in cases of typhoid 
infection is reported by Griinbaum, 1 who notes that he obtained a 
positive reaction in cases of febrile jaundice ; he suggests that in 
these cases the infection was caused by one of the intermediate 
organisms between the typhoid bacillus and the bacillus coli com- 
munis. In so-called paratyphoid fever, however, a positive Widal 
reaction is either absent or imperfect and only observed with a low 
dilution (see page 170). 

1 Griinbaum, cited by Durbam, Brit. Med. Jour., 1898, vol. ii, p. 600. 



168 THE BLOOD. 

WidaVs test is a most valuable aid in the diagnosis of typhoid fever, 
but cannot be relied upon to the exclusion of other symptoms. 

Technique. — The method is based upon the fact that typhoid 
serum will cause arrest of motility and agglutination of the specific 
bacilli even when diluted, whereas clumping of the same organism is 
obtained only with sera from other diseases and healthy individuals 
when these are used in a more concentrated form. The time-limit 
at which clumping occurs is likewise an important factor, as non- 
typhoid sera are at times met with in which, notwithstanding a cer- 
tain degree of dilution, agglutination occurs, providing that the speci- 
men is kept for a long time. Both factors, viz., the degree of dilu- 
tion necessary to eliminate the agglutinating power of non-typhoid 
sera, as also the time-limit of observation, have been arbitrarily de- 
termined. Widal originally advised a dilution of 1 : 10, and Griiber 
a time-limit of one-half hour. At the present time there is a ten- 
dency, among German physicians especially, to increase the degree 
of dilution to 1 : 40, and even 1 : 50, and the time-limit to from one 
to two hours. Generally speaking, a positive reaction is of greater 
value the greater the degree of dilution at which it can still be 
obtained. A uniform standard, however, is necessary in order to 
allow a strict comparison of results, and I am personally inclined 
to favor the German standard. The degeee of dilution should exceed 
1 : 40, as undoubted positive reactions in non-typhoid individuals 
have been obtained with 1 : 20 and even 1 : 40. 

In any event, only a full-virulent, fresh bouillon culture of the 
typhoid bacillus, viz., one not older than sixteen to twenty-four 
hours, should be used. The further technique is simple : 1 volume 
of blood-serum is diluted with the requisite amount of the bouillon 
culture, viz., to 10, 20, 30, 40, or 50 volumes, as the standard may 
be. Of this mixture, one drop is mounted on a slide, covered, and 
examined with a moderately high power. If the case in question 
is one of typhoid fever, it will be observed that after a variable 
length of time the individual bacilli, which at first actively dart 
about the field of vision, become quiescent and tend to gather in 
distinct clumps, while the interspaces become entirely free from ba- 
cilli or very nearly so. After one-half hour, or one or two hours, 
according to the degree of dilution, all motion has ceased. When 
the time-limit has expired and loss of motility and agglutination 
have not occurred the result is negative. In such an event further 
examinations should be made on the following days. In every case 
it is well to make a control-test with the simple bouillon culture, so 
as to insure the absence of preformed clumps and the virulence of the 
organism ; of the latter, the degree of motility is the best index. 

In order to secure the necessary degree of dilution, various meth- 
ods have been suggested. The simplest and the one generally em- 
ployed in municipal bacteriological laboratories, is to receive a large 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 169 

drop of blood upon a slide or slip of glazed paper, and allow it 
to dry. A drop of distilled water is then placed on the blood and 
allowed to remain for several minutes, when it is washed off and 
intimately mixed with the requisite number of drops of the bouillon 
culture, and examined as described. The principal advantages of 
this method are its simplicity and the fact that the dried blood 
retains its agglutinating properties for weeks and months. The 
results, however, are less reliable than with the use of liquid blood. 
If this is to be employed, properly graduated capillary pipettes are 
prepared, similar to the pipettes accompanying the Thoma-Zeiss 
hsemocytometer. Blood is first drawn up to a given mark and 
expelled into a small watch-crystal ; the requisite amount of the 
bouillon culture is then obtained with the same pipette and imme- 
diately mixed with the blood, and a drop of the mixture is ex- 
amined under the microscope. Sterilization of the apparatus used 
is unnecessary, and each pipette is destroyed after use. 

If it is desired to keep the liquid blood for any length of time, 
similar pipettes may be used with a small bulb blown in the middle. 
These are first sterilized by heat and sealed at the ends. Before use, 
one end is broken off, the bulb heated in a spirit flame, and filled by 
capillary attraction. It is then again sealed, when the blood may 
be kept indefinitely. Another method, which is said to be even 
more reliable than those mentioned, is the following : 

After careful disinfection of the arm, 5 or 6 c.c. of blood are 
withdrawn from one of the superficial veins, by means of a sterilized 
hypodermic syringe, and placed in a sterilized test-tube measuring 
from 10 to 12 cm. in length. The blood is allowed to stand until 
the serum has separated from the clot, which may be hastened by 
loosening the coagulum from the walls of the tube with a platinum 
needle. Eight drops of the serum are added to 4 c.c. of nutrient 
bouillon, which should be as nearly neutral as possible, when the 
mixture is inoculated with 1 oese (platinum loopful) of a fresh 
bouillon culture of the typhoid bacillus not more than twenty-four 
hours old. The tube is kept at a temperature of 37° C. for twenty- 
four hours. At the end of this time, and frequently earlier, the 
bouillon will be absolutely clear, or very nearly so, while little flakes, 
composed of the bacilli, will be seen at the bottom and adhering to 
the sides of the tube, if the case under observation is one of typhoid 
fever; otherwise the bouillon becomes uniformly cloudy and a 
true sediment is not formed. A pseudo-reaction also may occur at 
times, which should not be confounded with the one just described. 
Innumerable microscopical, dust-like particles will then be seen 
scattered throughout the fluid, which can readily be distinguished 
from the cloudy appearance of non-typhoid specimens. It has been 
suggested that this result is obtained in cases of intense infection 
with the Bacillus coli communis. Should doubt arise, it is only 



170 THE BLOOD. 

necessary to keep such tabes for a few hours at a temperature of 
37° C, when it will be noticed that the dust-like aspect has given 
place to the ordinary cloudy appearance observed in cases which are 
not typhoid fever. 

Of the nature of the substance or substances which cause agglu- 
tination — agglutinins — little is known that is definite. It appears 
that in the blood they are intimately associated with fibrinogen and 
globulin, as plasma from which these two bodies have been removed 
no longer possesses agglutinating properties. As chemical differ- 
ences, however, apparently do not exist between normal globulin 
and globulin obtained from typhoid blood, it seems likely that the 
substances in question do not form an integral part of the globulin 
molecule, but perhaps are thrown down mechanically when the 
proteid substances are precipitated. This view is rendered probable 
by the fact that typhoid urine free from albumin may likewise cause 
arrest of motility and agglutination of typhoid bacilli. Attempts to 
separate the agglutinins from the proteids of the blood have thus 
far not been successful. 

The milk of immunized animals or of typhoid patients acts like 
the blood, and in it the agglutinins are apparently associated with 
casein. Exposure of such milk to a temperature of 80° C. destroys 
its agglutinating power. Very interesting is the observation of 
Malvoz, that very dilute solutions of safranin and vesuvin act upon 
the typhoid bacilli as typhoid serum does, and upon these bacilli only. 

Literature. — Pfeiffer, Zeit. f. Hyg., vol. xviii. p. 1. Pfeiffer u. Kolb, Deutsch. 
med. Woch., 1896, p. 185. Griiber u. Durham, Munch, med. Woch., 1896, pp. 206 
and 285. Widal, Soc. med. des Hop., 1896, p.. 561 ; and Presse med., 1897, i. p. c. Biggs 
and Park, Am. Jour. Med. Sci.. vol. cxiii. p. 274. Stewart, Trans. Am. Pub. Health 
Assoc., vol. xxiii. p. 151. Forster, Zeit. f. Hyg., vol. xxiv. p. 500. Da Costa, N. Y. 
Med. Jour., 1897. Anders and McFarland, Phila. Med. Jour., 1899, pp. 778 and 832. 
Bieberstein (collective work), Zeit. f. Hyg., 1898, vol. xxvii. Tobiesen (350 cases), 
Zeit. f. klin. Med., 1901, vol. xliii. p. 147. 

Paratyphoid Fever. 

In cases of so-called paratyphoid fever organisms may be met 
with in the blood which apparently occupy a position intermediate 
between the typhoid bacillus and the organisms belonging to the colon 
group. Collectively they are spoken of as paratyphoid or paracolon 
bacilli, though the question whether or not they represent a well- 
defined species has not been definitely settled. 

Cases of paratyphoid fever clinically resemble true typhoid, but 
their serum does not react with the typhoid bacillus, or at least only 
imperfectly so and with a low dilution, while the organism which 
appears to be pathogenic in the individual case is agglutinated in a 
typical manner. Unfortunately, however, the serum of one case will 
not always react with the organism of a second case, so that it seems 
rather doubtful if the serum reaction will prove of value in distin- 
guishing the intermediates as an entire group from typhoid on the 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 171 

one hand, and the bacillus coli on the other. Many varieties appar- 
ently exist (Gwyn's paracolon bacillus, Cushing's bacillus 0, Hew- 
lett's bacillus b, Noonan's bacillus, etc.). 

Organisms belonging to this group have been described by Widal, 
Gwyn, dishing, Schottmuller, Kurth, Brill, Johnston, Coleman- 
Buxton, and others. It is likely that the small percentage of cases 
which are clinically typhoid fever, but in which the Widal reaction 
is persistently absent, are cases of this order. 

The examination of the blood is conducted as in typhoid fever, 
although it is perhaps not always necessary to dilute the blood to 
the same degree. In Gwyn's case successful cultivation followed the 
spreading of a few c.c. of blood over the surface of agar tubes or 
plates. Noteworthy is the fact that the intermediates do not form 
gas in lactose media ; in saccharose media also they produce no gas, 
though this fact is less important, as many true colon bacilli likewise 
are incapable of causing its fermentation. Milk is not coagulated. 
Schottmuller states that the organisms at first render the milk acid, 
but that subsequently the reaction becomes alkaline. On potato the 
growth is slight and there is no discoloration. 

Literature. — Gwyn, Johns Hopkins Hosp. Bull., 1898, vol. ix. p. 54. Cashing, 
Ibid., 1900, vol. xi. p. 156. Schottmuller, Zeit. f. Hyg., 1901, vol. xxxviii. W. B. 
Johnston, Am. Jour. Med. Sci., 1902, vol. cxxiii. p. 187 (analysis of all cases reported 
up to that time). A. W. Hewlett, Ibid., p. 200. Coleman -Buxton, Ibid., 1903, vol. 
cxxiii. p. 976. See also Ascoli, Zeit. f. klin. Med., 1903, vol. xlviii. p. 419. 

Pneumonia. 

Recent research has brought to light the interesting fact that in 
fatal cases of acute croupous pneumonia the specific diplococcus is 
quite frequently present in the blood, while in cases ending in recov- 
ery it is encountered only exceptionally. I have found, as a matter 
of fact, that a positive result is obtained in more than 50 per cent, 
of the fatal cases. The invasion of the blood usually occurs twenty- 
four to forty-eight hours before death, but may take place at an earlier 
date or be delayed. From the standpoint of prognosis a bacterio- 
logical examination of the blood may thus be of considerable impor- 
tance. It should be remembered, however, that while a positive 
result is always a symptom mail ominis, there are cases on record in 
which recovery occurred notwithstanding the presence of diplococci 
in the blood. In such cases metastatic infection probably has oc- 
curred. 

Prochaska, working under Eichhorst's direction, reports that he 
found pneumococci in the blood in each of 10 cases examined, and 
in a subsequent series of 40 cases, of which 7 were fatal, he obtained 
the pneumococcus in 38. Twice there developed organisms concern- 
ing the nature of which the author is uncertain. These were strepto- 
cocci with a tendency to arrangement in pairs, and may have been an 



172 THE BLOOD. 

especially virulent variety of pneumococcus. One of these cases ended 
fatally ; in the second slow resolution was followed by a secondary 
indurative process. The cultures were made with 10 c.c. of blood, 
at varying periods of the disease, sometimes as early as the second 
day. It is noteworthy that in two cases positive results were found 
on the day following the crisis ; in one, followed by empyema, on the 
second day after the crisis ; and in another case, in which slow reso- 
lution was taking place, organisms were found three days after the 
febrile crisis. 

Cole examined 30 cases at the Johns Hopkins Hospital and ob- 
tained positive results in 9. All of these ended fatally, but it is to 
be noted that 4 additional cases of the series died, and that in these 
the pneumococcus was not obtained. 

Frankel states that according to his experience, which is based 
upon an examination of more than 150 cases, one may infer that 
death will occur either with the symptoms of sepsis or that metas- 
tasis will take place in the internal organs whenever a larger number 
of colonies develop on spreading 1 c.c. of blood upon a plate of 
agar. If, however, the number is so small that it is necessary to 
take larger amounts of blood to demonstrate their presence, and to 
grow them in bouillon instead of on agar, so as to eliminate the bac- 
tericidal power of the blood altogether, then Frankel believes their 
presence is of no significance, and does not warrant a fatal progno- 
sis. In the latter case he has found that the bacteria are frequently 
avirulent. 

Of 72 cases of pneumonia which came to autopsy at the Boston 
City Hospital in a total of 341 deaths from the disease, the pneumo- 
coccus was found in the blood in 36, either alone or together with 
other organisms. No ante-mortem cultures, however, were made. 
These negative results, in the face of the positive findings of Pro- 
chaska, are no doubt referable to some difference in technique. 

The examination, which should be repeated every day, is conducted 
as follows : After disinfection of the arm one of the superficial veins 
is compressed with a finger and punctured with an ordinary hypo- 
dermic syringe which has previously been sterilized in boiling water. 
10 c.c. of blood are aspirated and agar tubes — liquefies at 40° C. — 
inoculated, each with 1 or more c.c. of the blood. Plates are then 
prepared and kept at a temperature of from 35° to 37° C. The 
colonies number from 2 to 200, and appear as small round, grayish, 
jelly-like drops, which are quite characteristic. During their growth 
they cause a greenish discoloration of the blood-agar. Other bacteria 
possess the same property, but to a less marked degree than the diplo- 
coccus pneumoniae. 

Instead of agar, bouillon may also be employed, and it is quite 
likely, as Prochaska suggests, that in this manner positive results 
may be more frequently obtained. Cole recommends the use of 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 173 

sterile litmus milk, of which portions of 150 c.c. each are employed 
in Erlenmeyer flasks. Early acidification and coagulation occur, 
and it is thus possible to determine more readily and quickly whether 
growth has taken place. Smears are then made and examined for 
capsules (see below). The identity is established by the characteristic 
shape and staining reactions of the organism, including the staining 
of the capsules, by the typical growth in milk and agar, and by the 
absence of growth, or very slight growth, in gelatin at ordinary room 
temperature. 

The individual organism (Plate XVIL, Fig. 2) is capsulated, and 
usually occurs in pairs, arranged end to end or in short chains. At 
times, however, the chains are quite long, and then it may be difficult 
to distinguish it from streptococci. It is easily stained with the com- 
mon anilin dyes. In order to differentiate the capsule, the following 
method, suggested by Welch, is best employed : Spread and dried 
cover-glass preparations are treated first with glacial acetic acid, 
which is allowed to drain off, and is replaced (without washing in 
water) with anilin-gentian-violet solution. The staining solution is 
added repeatedly until all the acid is replaced. The specimen is 
now washed in a weak salt solution (about 2 per cent.) and examined 
in this, and not in balsam. The capsule and coccus can thus be dif- 
ferentiated. 

Literature.— Goldscheider, Deutsch. med. Woch., 1892, No. 14. Sittmann, 
Deutsch. Arch. f. klin. Med., 1894, vol. liii. p. 323. Kiihnau, Zeit. f. Hyg., 1897, vol. 
xxv. Kohn, Deutsch. rned. Woch., 1897, p. 136. James and Tuttle, N. Y. Presby- 
terian Hosp. Eep., vol. iii. p. 44. Sello, Zeit. f. klin. Med., 1898, vol. xxxvi. White, 
Jour, of Exper. Med., 1899, vol. ii. Silvestrini and Sertoli, Eiforma Med., 1899, No. 
116. Abstr. in Centralbl. f. inn. Med., 1899, vol. xxi. E. Cole, Johns Hopkins Hosp. 
Bull., 1902, vol. xiii. p. 236. Prochaska, Centralbl. f. gen. Med., 1900, No. 46. Pro- 
chaska, Deutsch. Arch. f. klin. Med., vol. lxx. p. 559. Frankel, Deutsch. med. Woch., 
1901, V. B., p. 212. 

Sepsis. 

The importance of a careful bacteriological examination of the 
blood in septic conditions has been definitely established. The 
technique is the same as that described above (page 169). But 
whether or not it is always necessary to use so much blood as in 
typhoid fever and pneumonia and so high a degree of dilution, may 
be questioned. The media which are commonly employed are the 
ordinary laboratory media ; in addition Libman has suggested the 
use of serum-glucose-agar and serum-glucose-bouillon. He has 
pointed out that on these media the growth of most bacteria is much 
more marked and more rapid than on ordinary serum-agar. This 
is true especially of the streptococcus, the pneumococcus, the gono- 
coccus, and the meningococcus. 

Petruschky has shown that in severe cases of septic infection it is 
almost always possible to find streptococci in the blood, while in the 



174 THE BLOOD. 

milder cases a negative result is usually reached. He has found, 
moreover, that while as a rule the presence of streptococci will justify 
a grave prognosis quoad vitam, death does not necessarily occur in 
every case. Other investigators have arrived at similar conclusions. 
Petruschky's positive findings include 5 cases of sepsis following 
phlegmonous abscess or pneumonic infection, with 3 deaths and 2 
recoveries ; 9 cases of puerperal infection, with 3 deaths and 6 recov- 
eries ; 1 case of ulcerative endocarditis (fatal termination) ; and 2 
cases of mixed infection with 1 death and 1 recovery. In 15 of 
the 1 7 cases streptococci were found ; in the 2 remaining cases 
staphylococci were present. 

Lenhartz obtained positive results intra vitam in 16 cases of endo- 
carditis out of 28. The organisms encountered were staphylococci, 
streptococci, the diplococcus pneumoniae, and in one instance the 
gonococcus. Most commonly a streptococcus parvus was found. 
Libman states that in cases of acute ulcerative endocarditis he has 
always found organisms in the blood ; he reports 2 instances, 1 acute 
and 1 chronic, in which the staphylococcus aureus was obtained in 
pure culture. Cole has recorded 1 instance of malignant endocar- 
ditis with septicaemia in which the staphylococcus albus was found. 
Libman reported a similar case, but later expressed the opinion that 
the organism may have been the staphylococcus aureus. He adds 
that in the last four years he had not met with a single instance in 
which he could ascribe a systemic infection to the staphylococcus 
albus. The same writer has further reported a series of 23 cases of 
systemic infection by the staphylococcus aureus, comprising a number 
of cases of osteomyelitis ; of these 23, 5 recovered. The amount of 
blood used in the examinations was usually from 5 to 15 c.c, some- 
times as much as 25 c.c. 1 

Pus organisms have been repeatedly found in the blood in phthisis, 
in advanced cases of the disease. F. Meyer and Michaelis state that 
they obtained positive results in 8 of 1 cases. Sittmann, Jacowsky, 
and Hewelke report similar findings, while others have been less 
successful, owing to the fact apparently that they used too little blood. 
Meyer and Michaelis suggest that it is not advisable to take less 
than 10 c.c. 

Hektoen has pointed out that in scarlatina streptococci may be 
found in the blood during life in at least 1 8 per cent, of all cases ; 
I append his conclusions : Streptococci may occasionally be found 
in the blood of scarlet fever cases that run a short, mild, and uncompli- 
cated clinical course. They occur with relatively greater frequency 
in the more severe and protracted cases of the disease, in which there 

1 In a recent communication Libman tells me that he found attenuated streptococci 
in five cases of mild acute endocarditis following what clinically appeared to be 
typical articular rheumatism. They could be demonstrated during extended periods 
of time. He also notes that he has recently observed a case of systemic infection by 
the staphylococcus citreus (the first instance of its kind in his series). 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD 175 

may also develop local complications and clinical signs of general 
infection, such as joint-inflammations ; but even in the grave cases 
of this kind spontaneous recovery may take place. In fatal cases 
streptococci may not be demonstrable. The theory that scarlet fever 
is a streptococcus disease thus does not seem to receive direct support 
from these investigations. 

In diphtheria, measles, and smallpox infection with streptococci 
is also not uncommon. Other organisms may, however, also be met 
with, such as the various staphylococci, and quite commonly also, 
according to Jehle, the bacillus of influenza. He found the organ- 
ism in question in 22 cases of scarlatina out of 48 that ended fatally ; 
in measles 15 times out of 23 ; and in 5 cases of varicella out of 9. 
In Hektoen's series, on the other hand, the organism was not found ; 
but it is noted that during the year influenza was not especially 
prevalent in Chicago. In the only 2 fatal cases of Hektoen's the 
staphylococcus aureus was found, and no streptococci. 

Of other organisms which may be met with in septic conditions, 
the diplococcus pneumonias is the most common. Aside from pneu- 
monia,, it has been found in peritonitis, associated with carcinoma 
of the uterus, in cases of suppurative oophoritis, following childbirth, 
in cases of biliary abscess at the time of the chill, etc. Friedlander's 
bacillus has also been found. In several cases of gonorrhoea! septi- 
caemia the gonococcus has been isolated during life (see below). 
Proteus vulgaris has been found in a few instances. The bacillus 
aerogenes capsulatus, which is so frequently seen after death, has also 
been obtained from the blood of living patients. Quite recently 
also a newly discovered micro-organism has been isolated from the 
blood by MacCallum and Hastings, which they term the micrococcus 
zymogenes. It is apparently closely related to the pneumococcus 
and the streptococcus pyogenes. 

The number of organisms which may be found in the blood in 
septic conditions is exceedingly variable. On the one hand, but one 
plate or flask out of several may show any growth, and then only 
after several days ; while, on the other hand, the number of organ- 
isms may be quite large. Cole has reported a case of streptococcus 
septicaemia in which the number of organisms amounted to 3642 
per c.c. of blood six days before death, and then rose to 10,716 per 
c.c. two days before death. 

The time before death at which organisms may be found in the 
blood is also quite variable ; sometimes they may be demonstrable 
a month before, in other cases only a day or two before the fatal 
issue. 

The Staphylococcus pyogenes aureus occurs in the form of spheri- 
cal bodies, averaging about 0.8 p. in diameter, which readily stain 
with the basic anilin dyes, as also with Gram's method. They 
usually occur in clumps, but may also be seen in pairs and in short 



176 THE BLOOD. 

chains. The organism grows on all culture-media, and in the pres- 
ence of oxygen gives rise to the formation of an orange-yellow pig- 
ment. Gelatin is rapidly liquefied ; it coagulates milk and clouds 
bouillon. The Staphylococcus pyogenes albus and citreus differ from 
the aureus by the absence of pigment in the first and by the forma- 
tion of a lemon-yellow pigment in the second. 

The Streptococcus pyogenes (Plate VII., Fig. 1) occurs in chains 
of spherical cocci which usually vary from four to twenty in number. 
The size of the individual organism is somewhat greater than that 
of the staphylococcus, but may vary even in one and the same chain. 
It is readily stained with the basic anilin dyes and also with Gram's 
method. It grows on all culture-media at the temperature of the 
room, forming small gray granular colonies on agar and gelatin. As 
a rule, it does not liquefy gelatin, and it may or may not coagulate 
milk and cloud bouillon. Several varieties are recognized, viz., 
Streptococcus brevis, which forms short chains ; Streptococcus longus, 
which occurs in long chains ; streptococci which render bouillon 
cloudy, and those which do not.; streptococci which form nocculent, 
sandy, scaly, or viscous sediments. 

The Streptococcus conglomeratus grows, without clouding bouillon, 
in the form of dense separate particles, scales, or thin membranes 
at the bottom and sides of the tube, and on shaking the sediment 
it breaks up into little specks, without producing uniform, diffuse 
cloudiness. The chains are long and interwoven in conglomerate 
masses (Welch). 

Literature. — F. W. White, " Cultures from the Blood in Septicaemia, Pneumonia, 
Meningitis, and Chronic Diseases," Jour. Exper. Med., vol. iv. p. 425. Petruschky, 
Zeit. f. Hyg., vol. xvii. p. 59. Sittmann, Deutsch. Arch. f. klin. Med., vol. liii. 
p. 323. Canon, Deutsch. Zeit. f. Chir., vol. xxxiii. p. 571 ; and Mitth. aus d. Grenz- 
geb. d. Med. u. Chir., 1902, vol. x. p. 41. Lenhartz, Munch, med. Woch., 1901. 
Nos. 28 and 29. Lihman, Proc. N. Y. Path. Soc, 1903, vol. iii. pp. 5 and 57; "On 
Certain Features of the Growth of Bacteria," etc., Jour. Med. Eesearch, 1901, vol. vi. 
Cole. Johns Hopkins Hosp. Bull., 1902, vol. xiii. p. 252. Wm. Welch, "Morbid 
Conditions Caused by the Bacillus Aerogenes Capsulatus," Ibid., 1899, vol. x. p. 134. 
Gwyn, Ibid., 1900, vol. xi. p. 185 (first case) ; Cole, Ibid., 1902, vol. xiii. p. 234 (sec- 
ond case). Hektoen, Jour. Am. Med. Assoc, 1903, vol. xl. No. 11. Jehle, Zeit. f. 
Heilk., 1901, vol. xxii. p. 190. Ewing, Trans. Am. Assoc. Phys., 1902, vol. xvii. p. 
208. 

Gonococcus Septicaemia. 

Cases of gonorrheal septicaemia in which the gonococcus was 
isolated from the blood of the patients during life have been re- 
ported by Thayer-Blumer, Thayer-Lazear, Byelogoway, Wilson, and 
Harris- Johnston. In all these cases gonorrheal endocarditis existed. 
In other infections of the same nature positive results were obtained 
by Ahmann, Colombini, Panichi, and Unger, in association with 
polyarthritis, epididymitis, myositis, tenovaginitis, inguinal bubo, and 
parotitis. In the endocarditis cases cultures were obtained after an 
illness lasting for from five weeks to seven months, at times as early 



PLATE X 



FIG. 1. 



FIG. 3. 



Streptoeoeeus Pyogenes. (Abbott.) 
FIG. 2. 




S\ 



Bacillus Anthraeis, bigbly magni- 
fied to show Swellings and Concavi- 
ties at extremities of the Single Cells. 
(Abbott.) 



o°§§e 






o 



<%9 



"^^ & 



Spirilla of Relapsing Fever, 
(v. Jaksch.) 



FIG. 4. 




L. 8CHMIDT, FEC. 



Malarial Blood Stained with Chenzinsky-Plehn's Solution. 
(Personal Observation.) 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. Ill 

as the ninth to the eleventh day preceding death, and on an average 
five days before death. 

To cultivate the gonococcus from the blood during life, it is 
neither necessary to use a large amount of blood nor to dilute it 
greatly, nor to employ any specially prepared medium. From 2 to 
5 c.c. are sufficient. According to Harris and Johnston, it is more 
advantageous to mix the blood with melted agar and to plate the same, 
than to use fluid media where the oxygen supply is more restricted. 

For a description of the organism, see page 644. 

Literature. — N. M. Harris and W. B. Johnston, "Gonorrhoeal Endocarditis with 
Cultivation of the Specific Organism from the Blood during Life," Johns Hopkins 
Hosp. Bull., 1902, vol. xiii. p. 236 (literature). Thayer and Blumer, Ibid., 1896, vol. 
vi. p. 59. Thayer and Lazear, Jour. Exper. Med., vol. iv. p.. 81. 

Anthrax. 

The bacillus of anthrax, as first pointed out by Pollender, Brouell, 
and Davaine, is frequently met with in the blood, where it should 
be sought for in doubtful cases by staining with Loffler's method. 
The number of the organisms present, however, is probably always 
small. Cover-glass preparations are floated for five to ten minutes 
on a mixture of 30 c.c. of a concentrated alcoholic solution of 
methylene-blue and 100 c.c. of a 1 : 10,000 solution of potassium 
hydrate; they are then washed for five to ten seconds in a 0.5 per 
cent, solution of acetic acid, treated with alcohol, dried, and 
mounted in balsam. Thus stained, the bacilli appear as rods meas- 
uring from 5 fx to 12 fi in length by 1 f± in breadth, and usually 
present a segmented appearance, the extremities being slightly thick- 
ened. Spores are not found, as the organism multiplies by fission. 
When present in large numbers it is not even necessary to stain, as 
the organisms can then be seen without difficulty in fresh specimens 
(Plate X., Fig. 2). 

In doubtful cases, in which a microscopical examination of the 
blood yields negative results, a few cubic centimeters of the blood 
may be injected into a mouse or a guinea-pig, in the blood of which 
the bacilli will soon be found in enormous numbers if the disease 
is anthrax. 

Literature. — Pollender, Casper's Vierteljahrsch. f gerichtl. u. offentl. Med., 1855, 
vol. viii. p. 103. Brauell, Virchow's Archiv, vol. xi. p. 132, and vol. xiv. p. 32. Da- 
vaine, Compt. rend, de l'acad. d. sci., vol. lvii. p. 220. Blumer and Young, Johns 
Hopkins Hosp. Bull., 1885, p. 127. 

Acute Miliary Tuberculosis. 

In acute miliary tuberculosis tubercle bacilli have repeatedly been 
observed in the blood ; but. while their presence may be regarded as 
pathognomonic of the disease, the search for them is most tedious 
and often in vain. Nevertheless a careful examination of the blood 
is indicated in doubtful cases ; but only a positive result is of value. 

12 



178 THE BLOOD. 

According to Liebmann, the tubercle bacilli are most numerous in 
the blood about- twenty-four hours after the injection of tuberculin. 
Working in this manner, he claims to have obtained positive results 
in 56 cases of 141. As a rule it is probably better to resort to the 
animal experiment. 

For methods of staining and a description of the tubercle bacillus, 
the reader is referred to the chapter on Sputum. 

Literature.— Liebmann, Berlin, klin. Woch., 1891, p. 393. Kronig, Deutsch. 
med. Woch., 1894, vol. v. p. 42. 

Glanders. 

In glanders the specific bacillus is constantly present in the blood, 
and may be demonstrated by staining the dried preparations on a 
cover-glass for five minutes with a concentrated alcoholic solution 
of methylene-blue, mixed with an equal volume of a 1 : 10,000 
solution of potassium hydrate just before using. From this mixture 
the specimen is passed for a second or two into a 1 per cent, solu- 
tion of acetic acid which has been tinged a faint yellow by the 
addition of a little tropreolin 00 solution ; it is then decolorized by 
washing in water containing 2 drops of concentrated sulphuric acid 

Fig. 34. 
• 



Bacillus of glanders. (Abbott.) 

and 1 drop of a 5 per cent, solution of oxalic acid for each 10 c.c. 
In specimens thus stained, the bacilli appear as short rods measur- 
ing from 2 fi to 3 [i in length by 0.3 fi to 0.4 fx in breadth, often 
containing a spore at one end (Fig. 35). 

Literature. — Duval, Arch, demed. exper., 1896, p. 361. 

Influenza. 

In the sputum of influenza a specific organism has been described 
by Pfeiffer and Kitasato ; it is said to be constantly present also in 
the blood of such patients. The organism in question appears in 
the form of minute rods measuring 0.1 ju in breadth by 0.5 ju in 
length occurring either singly or in chains of three or four. In 
suitably prepared specimens, owing to the fact that their poles take 
up the stain more readily than the middle portion, they convey the 
impression of diplococci. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 179 

Canon advises the following method for demonstrating their pres- 
ence in the blood : cover-glass preparations that have been allowed 
to dry at ordinary temperature are placed in absolute alcohol for 
five minutes and are then stained at a temperature of 37° C. for 
from three to six hours, with Chenzinsky-Plehn's solution (see page 
101). The specimens are washed in water, dried between layers of 
filter-paper, and mounted in balsam. Stained in this manner, the 
red corpuscles are colored red, and the leucocytes, as well as the 
bacilli, blue. As a rule, only from four to twenty are found in one 
preparation, usually occurring singly, but also in groups. Owing 
to the fact that they are found in the blood only during the acme 
of the disease, Canon recommends examination of the sputum for 
diagnostic purposes, a view with which my own observations are 
entirely in accord. Some observers indeed deny the occurrence of 
the organism in the blood altogether (Kuhnau). 

Literature. — Canon, Virchow's Archiv, vol. cxxxi. p. 401.. Klein, Baumgar- 
ten's Jahresb., 1893, p. 206. Kiihnau, Zeit. f. Hyg., vol. xxv. p. 492. 

Relapsing Fever. 

Relapsing fever is characterized by the presence in the blood, and 
here only, of spirilla or spirochete which bear the name of their 
discoverer, Obermeier. In order to search for these organisms no 
special precautions are necessary. After having carefully cleansed 
the finger, as described, a drop of blood is mounted on a very thin 
cover-glass. This is inverted directly upon the slide, when the 
specimen is ready for examination ; an oil-immersion lens is not 
required.. Attention is drawn to the presence of the organisms by 
certain disturbances which are noticeable among the red corpuscles, 
and upon careful examination it will be seen that these are caused by 
the wriggling movements of the spirilla. The Spirochete Obermeieri 
are long, slender filaments, measuring from 36 fi to 40 fi in length 
by 0.3 /z to 0.5 /z in breadth, and present from eight to twelve 
incurvations of equal size with tapering extremities (Plate X., 
Fig. 3). These two last characteristics serve to distinguish this 
species from that described by Ehrenberg, in which the radius of the 
incurvations is not the same in all, and in which the extremities do 
not taper. 

The number of spirilla which may be found in a drop of blood 
varies, being greater during the access of the fever, when twenty, or 
even more, may be observed in the field of the microscope. They 
occur singly or in bunches of from four to twenty, specimens resem- 
bling those figured in the illustration being frequently seen. In the 
quiescent stage they are arranged sometimes in the form of rings or 
of the figure 8. After the crisis they seem to disappear entirely, and 
their presence during an afebrile period may therefore be regarded as 
indicating a pseudocrisis. During the afebrile periods small, bright, 



180 THE BLOOD. 

round bodies have been described as occurring in the blood, which 
according to some are spores, but according to others represent merely 
debris of the spirilla. 

Culture-experiments have not been very satisfactory, although 
Koch observed an increase in their number at a temperature of from 
10° to 11° C. 

That confusion should ever arise in distinguishing the spirilla of 
relapsing fever from the free flagella observed at times in malarial 
blood seems to me very improbable. 

Literature. — Heidenreich. Untersuch. iiber d. Parasit. d. Biiekfallstyphus, Ber- 
lin, 1877. Moczutkowsky, Deutsch. Arch. f. klin. Med., vol. xxiv. p. 80, and vol. xxx. 
p. 165. Blisener, Inaug. Diss., Berlin, 1873. Engel, Berlin, klin. Woch., 1873, p. 409. 

Malta Fever. 

In Mediterranean or Malta fever the specific organism, the 
Micrococcus melitensis (Bruce), has been isolated from the blood 
during life, but as a rule the findings are uncertain. Diagnosis 
is facilitated by the fact that a well-pronounced agglutination is 
obtained with the patient's serum. A positive reaction with a 
dilution of more than 1 : 20, according to Birt and Lamb, may be 
regarded as proof positive of the existence of the disease. As a 
rule agglutination can still be obtained with a dilution of from 
1 : 600 to 1 : 700. It begins about the fifth day of the disease, and 
gradually diminishes in intensity during convalescence, but may 
persist for a year and a half, and even longer. 

The organism in question is a coccus, measuring 0.3 p. in diam- 
eter, and occurs singly, in pairs, and sometimes in fours. Longer 
chains are not seen. It is motile. It is stained by the usual dyes 
and grows best on 1.5 per cent, of very feebly alkaline, peptonized 
agar-beef jelly. After thirty-six hours the colonies are a transparent 
amber, while later they are opaque. Liquefaction does not occur. 

Literature.— C. Birt and G. Lamb, " Mediterranean Fever," Lancet, Sept. 9, 
1899. Wright and Smith, Brit. Med. Jour., April 10, 1897. Musser and Sailer, Phila. 
Med. Jour., 1898, p. 1408. and 1899, p. 89. E. P. Strong and W. E. Musgrave, 
" The Occurrence of Malta Fever in Manila," Phila. Med. Jour., 1900, p. 996. J. J. 
Curry, " Malta Fever," Jour. Med. Besearch, July, 1901. 

Yellow Fever. 

"Wasdin and Geddings, constituting a commission of medical 
officers of the U. S. Marine-Hospital Service detailed by the U. S. 
government to investigate the cause of yellow fever, report that 
Sanarelli's bacillus may be isolated from the blood of the patients 
during life. They found the organism in twelve cases out of four- 
teen after the third day of the disease, and also obtained it from 
the remaining two after death. In other diseases it was not found. 

A similar commission, consisting of Reed, Carroll, Agramonte, 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 181 

and Lazear, on the other hand, arrive at negative results. By 
withdrawing the blood from the veins of nineteen patients they 
failed to obtain a positive result in every instance. Post-mortem 
investigations in eleven cases were likewise negative. 

According to Reed and Carroll, Sanarelli's Bacillus icteroi'des 
should be considered a variety of the hog cholera bacillus, and as a 
secondary invader in yellow fever. 

Infection occurs through the bite of mosquitoes (Culex fasciatus, 
Fabr., and probably other varieties also) which have previously fed 
on the blood of yellow fever patients. The period after contamina- 
tion which must elapse before the mosquito is capable of conveying 
the infection averages twelve days in summer, and eighteen or more 
days during the winter months. 

Literature.—" Controversy between G. Sanarelli and W. Eeed and J. Carroll on 
the Specific Cause of Yellow Fever," Med. News, 1899, pp. 193, 321, 513, and 737. E. 
Wasdin and H. D. Geddings, Eeport of Commission of Medical Officers to Investigate 
the Cause of Yellow Fever, Treasury Dept., U. S. Marine-Hospital Service, 1899. 
Eeed, Carroll, and Agramonte, Jour. Am. Med. Assoc, 1901, p. 431. 

Bubonic Plague. 

In advanced cases of bubonic septicaemia the specific organism 
may be found in the blood in small numbers. Toward the end of 
rapidly fatal cases they become more numerous, and may then be 
demonstrable directly with the microscope. 

The organism in question, the Bacillus pestls (Kitasato, Yersin), 
is a short, thick cocco-bacillus, with rounded ends, measuriug about 
2 p. in its greatest diameter. Examined in the hanging drop it is 
devoid of automobility. The polar regions are readily stained, 
while the interpolar area remains colorless. In many organisms a 
capsule can be made out by appropriate methods, but it is appar- 
ently not a constant feature. Oftentimes the form of the organism 
deviates from the normal. It may thus resemble a coccus on the 
one hand, while on the other it appears more elongated. It is 
decolorized by Gram's method. 

The blood-smears are fixed by immersion in absolute alcohol 
for twenty-five minutes ; or they are covered with absolute alcohol 
for about one-half minute, when the alcohol is burned off. For 
staining purposes, borax methylene-blue (a solution of 2 per cent, 
methylene-blue in 5 per cent, borax-water) or Loffler's alkaline 
methylene-blue may be conveniently employed. . In the first in- 
stance we stain for one-half minute, in the second for from two to 
three minutes. The polar staining is in this manner quite satis- 
factory. 

On gelatin-agar containing 2.5-3.5 per cent, of salt and in 
bouillon a fairly characteristic growth results. In the case of the 
agar involution-forms are obtained, among which long, slender 



182 THE BLOOD. 

bacilli, which are segmented and present a vacuolated appearance, 
are especially noteworthy. In this state they stain quite badly and 
have lost a certain degree of their virulence. In bouillon the or- 
ganism often forms long chains of well-rounded bodies which are 
quite similar to a coccus. During its growth in bouillon it forms 
flakes or flocculi, which rapidly sink to the bottom of the tube, 
leaving the liquid clear above. Colonies on gelatin about thirty- 
six hours old are warty, strongly refractive formations, which often 
present a delicate, irregularly indented margin. Even after twenty- 
four hours one can obtain smears, in which 50-100 bacilli are grouped 
in little colonies of irregular" form, while examination of the plates 
with a magnifying power of 60 diameters reveals scarcely any growth. 
The organism does not liquefy the gelatin. The optimum tempera- 
ture for growth is between 36° and 39° C. 

Literature. — For Kitasato's report see : Annual Eep. of the U. S. Marine- 
Hospital Service for 1894; W. Wyman, Bubonic Plague; U.S. Treasury Dept., 1900. 
Kossel u. Overbeck, Arb. aus d. Kais. Gesundheitsamt., 1901, vol. xviii. 

v 

Malaria. 

The discovery in the blood of a specific micro-organism belonging 
to the class of protozoa, the Plasmodium malarias of Laveran, and 
its invariable presence in the different forms of this disease, must be 
regarded as one of the most important in clinical medicine. This is 
not the place to state how frequently a diagnosis of malarial fever 
based upon clinical symptoms alone has proved false, nor how often a 
tubercular, a syphilitic, or a septic infection has been overlooked 
and termed malaria. It will suffice to say that errors of this 
kind, in view of our present knowledge and the ease with which 
they can be avoided by every physician, should no longer occur. 
The diagnosis of malaria should in every case be based upon a micro- 
scopical examination of the blood. The search for the specific organ- 
ism, it is true, may be very tedious at times, but it will always be 
crowned with success if the disease in question is malaria. 

The parasite in question, as I have stated, is a protozoon, and 
belongs to the class of hsematozoa, representatives of which are 
found in the blood of various animals, such as the rat, frog, turtle, 
carp, various birds, etc. Three varieties are known to occur in the 
blood of man, viz., the parasite of tertian, quartan, and sestivo- 
autumnal fever. The life-history of these organisms is now well 
understood, and it is known that in addition to the intra-corporeal 
cycle of development which takes place in the human body there is 
yet another, an extra-corporeal cycle, which occurs in certain mos- 
quitoes of the genus Anopheles. Infection occurs through the 
bites of such mosquitoes, which themselves have been infected by 
sucking the blood of malarial patients. This has been abundantly 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 183 

demonstrated by Ross, Maiison, Grassi, and others, and may be 
regarded as an established fact. 

Method of Examination. — The necessary amount of blood is 
best obtained by puncture of a finger or the lobe of the ear. Cover- 
glass specimens are then prepared as usual and may be examined 
either wet or stained with one of the modifications of the Roma- 
nowsky method, or the eosinate of methylene-blue (see page 127). 

Ross has recently suggested the advisability of spreading thick 
blood specimens and extracting the haemoglobin before staining for 
the malarial organisms, when these are only present in small num- 
bers. The search for the youngest forms of the sestivo-autumnal 
parasite especially is in this manner much facilitated. Ruge endorses 
this method in the following modification, but points out that the 
specimens are by no means beautiful owing to precipitation of 
pigments. A large drop of blood (about 20 cbmm.) is spread over 
a surface measuring about 18 square millimeters. The air-dried 
preparation is then placed for a few minutes in a 5 per cent, solution 
of formalin, 1 to which 0.5-1 per cent, of acetic acid has been added. 
In this manner the haemoglobin is all extracted, while at the same 
time the blood-film is fixed, so that it can now be washed without 
fear of ruining the preparation. This is then stained either accord- 
ing to one of the modifications of the Romanowsky method, or with 
the eosinate of methylene-blue, carbol-thionin, etc. Ruge further 
advises that specimens stained according to the Romanowsky method 
be subsequently stained with Manson's solution, 2 in order to render 
the smallest and medium-sized ring-forms more readily visible, as 
their affinity for the dye is somewhat impaired by the fixation in 
formalin. 

The Parasite. — The following forms of the parasite may be found 
in the blood : 

1 . Hyaline Non-pigmented Intracellular Bodies. — These 
apparently represent the earliest stage in the development of the 
parasite, and are found in all forms of malarial fever ; they are espe- 
cially abundant during the latter part of the paroxysm or immedi- 
ately thereafter. At first sight they may be mistaken for vacuoles, 
but upon closer examination it will be found that they exhibit dis- 
tinct movements of an amoeboid character, and may thus easily be 
recognized with a little experience. 

The rapidity with which these changes in the form of the organism 
occur in the tertian type of ague is most astonishing, and sketches 
of any one phase can often, indeed, be made only from memory ; 

1 This solution would contain 2 per cent, of formaldehyde gas, as the commercial 
formalin is about a 40 per cent, solution, 

2 This is an aqueous solution of borax (5 per cent.) and methylene-blue (2 per cent. ). 
The blood-films are stained with this solution for about thirty seconds ; they are then 
washed in water, dried with filter-paper, and afterward by gently warming them over 
the flame. 



184 THE BLOOD. 

in quartan fever the movements are much slower and far less exten- 
sive. 

In the irregular fever of the sestivo-autumnal form amoeboid 
movements may likewise be observed, but more commonly the para- 
site assumes a ring-like appearance, and does not throw out distinct 
pseudopodia. If these forms are carefully observed, however, it will 
be found that they are not absolutely quiescent, but alternately ex- 
pand and contract. 

In tertian fever the organism (Plate XI.) is pale and indis- 
tinct, while in quartan fever it is sharply outlined and somewhat 
refractive (Plate XII., Fig. 2). In the sestivo-autumnal form the 
organism is usually much smaller than in the tertian type, and the 
ring-like bodies frequently present at some point in their interior 
a distinctly shaded aspect which closely resembles the darker por- 
tion in the centre of a normal corpuscle (Plate XII., Fig. 1). It 
is thus possible, even at this stage in the development of the para- 
site, to distinguish between fever of the tertian, quartan, and sestivo- 
autumnal type. 

The numbers in which these small, non-pigmented intracellular 
organisms may at times be met with is most astonishing. In a case 
of pernicious malarial fever of the algid type, which I had occasion 
to examine, and in which a history of only one week's illness with- 
out chills was obtained, normal red corpuscles were indeed only 
exceptionally found. The case was one of the aestivo-autumnal 
form of fever. 

2. Pigmented Intracellular Organisms. — These represent 
a later stage in the development of the parasite, and, like the non- 
pigmented intracellular bodies, are met with in all types of malarial 
fever. Their appearance, however, differs considerably in the vari- 
ous forms. In tertian fever minute granules of a reddish-brown 
color appear in the bodies of the organism very soon after the par- 
oxysm. These gradually increase in number, while the invaded 
corpuscles proportionately become paler and paler, until finally only 
an indistinct, shell-like outline can be discerned. In fresh specimens 
the granules, which often assume the form of little rods, resembling 
bacteria, exhibit most active molecular movements, attracting atten- 
tion at once. The body of the parasite, which during its develop- 
ment has increased gradually in size, is probably hyaline, and may 
still be seen to undergo amoeboid movements. These are not nearly 
so active, however, as in the non-pigmented stage. The move- 
ments, moreover, cannot be followed so readily, owing to the pres- 
ence of the granules. At first sight, these appear to be scattered 
in small collections throughout the red corpuscle, and the impression 
may be gained that several organisms are present at the same time. 
Upon closer investigation, however, it will be seen that this is only 
apparently the case, and that the granules are confined to the bulb- 



PLATE XL 














L Schmidt fecit 



The Parasite of Tertian Fever. 



i, Normal Red Corpuscle; 2-4, Non-pigmented Stage of the Organism, showing Amoeboid Move- 
ments; 5-7, Progressive Pigmentation and Growth; 8-11, the Process of Segmentation; 12, Young 
Forms ; 13, Large Extra-cellular Organism ; 14, Mode of Formation of Extra-cellular Body ; 15, Small 
Fragmented Extra-cellular Organism ; 16, Flagellate Body and Free Flagella. Unstained Specimen. 
(Personal Observation.) 



PLATE XII. 



FIG. 1. 













A 



a oo a 



°o-^0 



*<f 




L Schmidt fecit 




The Parasite of Aestivo-Au.tu.mnal Fever, 
i, Normal Red Corpuscle; 2-10, Gradual Growth of the Organism ; n and 12, Segmenting Bodies; 
13, Young Forms; 14-22, Crescents, Ovoids and Spherical Bodies, with and without Bib; 23, Flagellate 
Body. Unstained Specimen. (Personal Observation.) 





Q 



FIG. 2. 







LSchmidt fecit 



The Parasite of Quartan Fever. 
1, Normal Red Corpuscle; 2-6, Gradual Growth of the Organism ; 7, Pigmented Extra-cellular Body ; 
8, Segmenting Body; 9, Young Forms; 10, Vacuolated Extra-cellular Body; 11, Flagellate Form. Un- 
stained Specimen. (Personal Observation.) 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 185 

cms extremities of the pseudo-podia of a single parasite. Before 
the end of forty-eight hours the organism has filled out the entire 
red corpuscle, which at the same time has attained a larger size 
than normal. The amceboid movements become less and less 
marked, and the pigment-granules, which may still be quite active, 
tend to collect about the periphery (Plate XL). 

In quartan fever pigmented intracellular bodies likewise appear 
very soon after the paroxysm. The individual granules, however, 
are somewhat larger, of more irregular size, and darker in color 
than those seen in the tertian type (Plate XII. , Fig. 2). Instead 
of exhibiting active molecular movements, moreover, they are 
almost entirely quiescent, and usually are grouped along the periph- 
ery of the organism. While amceboid movements can at first 
be observed, these become less and less marked, until finally, at 
the end of from sixty-four to seventy-two hours, they cease. 
The organism then presents a round or ovoid form, but does not 
fill the red corpuscle entirely. It is curious to note that in this 
form of ague the red corpuscles do not become decolorized, but 
rather darker than normally, and at times specimens may be seen 
which present a distinctly greenish or brassy appearance. When 
the parasite has become fully developed the corpuscle is smaller than 
normally, and, on staining, it may be seen that the organism still is 
surrounded by a narrow zone of corpuscular protoplasm even when 
this is not apparent in unstained preparations. 

The pigmented intracellular bodies which may be found in sestivo- 
autumnal fever (Plate XII., Fig. 1) can readily be distinguished 
from those observed in tertian and quartan ague. As in these 
types, pigment-granules also appear after the paroxysm ; they are 
never numerous, however, and often only one or two minute dark 
granules can be detected near the periphery. The organism, even 
in the later stages of its development, scarcely ever occupies much 
more than one-third of the corpuscle. Usually the granules exhibit 
scarcely any movements. As in the quartan type of ague, decolor- 
ization of the red corpuscles does not occur, and here, as there, a 
greenish, brassy appearance often is observed. At times the red 
corpuscles are shrunken, crenated, or spiculated. 

At the beginning and during the paroxysm forms are at times 
seen in which the few pigment-granules that may be present have 
gathered in the centre of the parasite and formed a solid clump. 
From the facts that these are observed only during the paroxysm, 
and that central blocks of pigment are found only during the stage 
of segmentation (see below) in tertian and quartan ague, Thayer 
and others conclude that these bodies are pre-segmenting forms of 
the parasite. This belief is strengthened further by the observation 
that pigment-bearing leucocytes are then also seen, which in the 
other types of fever likewise are found only at this time. 



186 THE BLOOD. 

3. Segmenting Bodies. — In cases of tertian and quartan fever the 
process of segmentation may be observed directly under the micro- 
scope, if specimens of blood are obtained just prior to or during the 
chill. In tertian fever organisms will then be seen in which the de- 
struction of the red corpuscles has advanced to a stage in which it is 
only possible to make out a pale contour of the original host. The 
parasite itself has assumed gradually a granular appearance, and the 
pigment-granules, which until then have exhibited pronounced mo- 
lecular movements, now become quiescent, larger and rounder, and 
show a distinct tendency to collect in the centre of the body. Here 
they form a roundish mass in which the individual components can 
scarcely be made out. While this change in the position of the pig- 
ment is taking place, beginning segmentation of the surrounding 
granular protoplasm will be observed. This at first is most marked 
at the periphery, from which delicate striae will gradually be seen to 
extend toward the central mass, dividing up the protoplasm into a 
number of oval bodies which closely resemble the petals of a flower 
(Plate XL). Still later these bodies, which in reality are the spor- 
ules of the parasite, will be found scattered in an irregular manner 
throughout the interior of the organism. The apparent envelope 
then disappears, and the sporules, which in tertian fever usually 
number from fifteen to twenty, lie free in the blood. Quite fre- 
quently, also, a sudden expulsion of the little bodies is observed and 
the impression gained as though the envelope had been burst asunder. 
Upon closer inspection, even at the petal stage, it will be seen that 
almost every sporule presents a tiny dot in its interior, which may 
at first sight be mistaken for a pigment-granule, but which in all 
probability is a nucleus. After the expulsion of the sporules these 
are frequently seen to move about in an active manner, but sooner 
or later they come to rest. 

While the progress of segmentation is very frequently observed to 
proceed in the manner described, this is not invariably the case. It 
may thus happen that segmentation occurs before the pigment- 
granules have had time to gather at the centre, or that the parasitic 
protoplasm breaks up into sporules directly without the intervention 
of the petal stage. In every case, however, the formation of sporules 
is associated directly with the occurrence of a paroxysm, and repre- 
sents the asexual type of reproduction of the parasite. 

The ultimate fate of the sporules is not definitely known, but it is 
likely that they in turn invade new corpuscles, cause their destruc- 
tion, and become segmented, thus giving rise to a new generation. 
As the process of segmentation, moreover, coincides in time with 
the occurrence of the chill, it is apparent that the interval elapsing 
between two consecutive chills — i. e., the type of the ague — depends 
upon the rapidity with which the non-pigmented forms arrive at 
maturity. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 187 

In quartan ague the manner in which segmentation takes place 
differs somewhat from that observed in the tertian form. It will 
here be observed that the pigment-granules, which have gathered 
along the periphery of the organism, as the parasite approaches ma- 
turity become arranged in a stellate manner, and apparently reach 
the centre through definite protoplasmic channels. Here they finally 
form a dense clump, and while the protoplasm assumes a finely 
granular appearance, segmentation proper begins and proceeds as 
in the tertian form. In quartan ague, however, the number of 
segments is smaller, varying between six and twelve. The entire 
segmenting body, moreover, is smaller than in the tertian form, and 
the segments are arranged in a more symmetrical manner. Here, 
indeed, the most perfect rosettes are observed (Plate XII., Fig. 2). 

In sestivo-autumnal fever segmenting bodies are only exception- 
ally seen in the peripheral blood, and it appears that the process of 
reproduction occurs principally in the spleen. The pre-segmenting 
forms described here undergo segmentation in a manner closely re- 
sembling that observed in tertian fever. The number of segments, 
moreover, is about the same, varying, as a rule, between ten and 
twenty. The segmenting body itself, however, is much smaller than 
in either the tertian or quartan form, and it is not possible to dis- 
tinguish any remains of the original host. 

4. Ckescentic, Ovoid, and Spherical Bodies (Plate XII., 
Fig. 1). — These are observed only in cases of sestivo-autumnal fever 
when this has persisted for at least one week. At first sight they 
apparently bear no relation to the other forms which have been 
described, and it has long been a question whether or not these 
bodies actually represent a stage in the life-history of the common 
malarial parasites. Grassi and Feletti have applied the name 
Laverania malarice to this form. More recent investigations have 
rendered it probable that they are derived directly from the pig- 
mented intracellular forms. Specimens may thus be met with in 
which' crescentic bodies are found in the interior of red corpuscles 
that have lost but little of their original color. Such observations, 
however, are not common. The typical crescents which are usually 
seen are highly refractive bodies, somewhat larger than a red cor- 
puscle, measuring from 7 /i to 9 p. in length by 2 jj. in breadth. 
Their extremities are usually rounded off and joined by a delicate, 
curved line bridging over their concave border. This is supposed 
to represent the remains of the original host. At other times this 
hood-like appendage is found along the convex border. The little 
pigment-granules and rods, which are always found in the interior 
of the crescents, are generally collected about the centre of the 
body, but they are occasionally also seen in one of the horns. While 
usually quiescent, a migration of some of the granules toward one 
extremity and back to the central mass may at times be observed. 



188 THE BLOOD. 

The ovoid and spherical bodies, which are usually much smaller than 
the crescents, exhibit the same general features, however, and often 
are provided likewise with a little hood. It is now known that 
the spherical bodies develop from the ovoids, and these again from 
the crescents. Like the crescents, the ovoid and spherical forms 
may be found in the interior of red corpuscles. 

5. Extracellular Pigmented Bodies. — In tertian and quar- 
tan ague some of the pigmented intracellular bodies, instead of 
undergoing segmentation when they have arrived at maturity, may 
be seen to leave their hosts and to appear as such in the blood. At 
the same time they increase considerably in size, and in the tertian 
form may indeed become as large as a polynuclear leucocyte (Plate 
XL). The pigment-granules, moreover, exhibit an activity in 
their movements which is most astonishing and never observed 
under other conditions. The outline of the parasite is then usually 
irregular and quite indistinct. Upon careful observation it will be 
seen that in some of these bodies the movements of the granules 
after a while become less and less marked, and finally cease, while 
the body of the parasite itself becomes still more irregular in out- 
line. This appearance is undoubtedly referable to the death of the 
organism. In others a gradual fragmentation is observed, small 
particles of the pigmented mother-substance being cut off from the 
parent-form. It is thus quite common to see the original parasite 
break up into four or five smaller bodies, in which the movements 
of the pigment-granules persist for some time. Sooner or later, 
however, even these cease, the outlines of the bodies become more 
and more indistinct, and death occurs. In still others the forma- 
tion of vacuoles may be observed, the pigment-granules at the same 
time becoming quiescent. This process is likewise regarded as one 
of degeneration. Most interesting, however, is the fact that flagel- 
lation may occur in some of these extracellular forms. It will then 
be observed that the pigment-granules which exhibit a most sur- 
prising activity tend to collect near the centre of the organism, 
while at the same time curious undulating movements may be made 
out along its contours. Suddenly one or more (one to six) extremely 
slender filaments will be seen to protrude from as many points on 
the periphery, presenting minute enlargements here and there in 
their course (Plate XL). The length of these filaments, or flagella, 
as they are termed, varies considerably. As a rule, it does not 
exceed the diameter of from five to eight red corpuscles, but much 
longer specimens are at times observed, and it appears to me that 
in most illustrations they are represented too short. With these 
flagella the organism makes most active whipping movements, scat- 
tering the red corpuscles to the right and left. Attention is, indeed, 
usually first drawn to the presence of these bodies by the disturb- 
ance which they cause in the field of vision. Occasionally one of 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD, 189 

the flagella may be seen to become detached from the body of the 
parasite and to move rapidly about among the corpuscles in a snake- 
like manner. In microscopical specimens they gradually come to 
a rest and often curl into a spiral. That difficulty should ever arise 
m distinguishing such detached flagella from the spirilla of relapsing 
fever seems very improbable, as the nature of these formations is 
shown by the presence or absence of other forms of the malarial 
organism. 

Beyond the fact that the flagellate organisms in tertian fever are 
larger than in the quartan form, no special points of difference exist 
(Plate XII., Fig. 2). 

In sestivo-autumnal fever similar changes may be observed. In 
crescents it is thus not at all uncommon to observe a small hyaline 
protrusion from the surface of the organism, which later may 
become detached. This process was formerly regarded as one of 
regeneration, but it is questionable whether this is actually the case. 
In other specimens, again, true fragmentation, or vacuolization, may 
occur, and flagellate bodies are met with in this type of fever as 
well as in tertian and quartan ague. The flagellates, as in quartan 
fever, are smaller than those observed in the tertian form, but other 
points of difference do not exist (Plate XII., Fig. 1). 

The significance of the flagellate organisms has until recently 
not been understood, but we now know that they represent the 
male element in the sexual reproduction of the malarial parasite, 
and the beginning of a cycle of development, which takes place 
outside of the human body, in the bodies of certain mosquitoes. 
The beginning of this cycle was observed first by MacCallum in the 
blood of infected crows. He here discovered that when one of the 
flagella broke loose it almost always sought out another full-grown 
form of the parasite which had not undergone segmentation, and 
penetrated this, just as the spermatozoon penetrates the ovum. 
Subsequently he observed the same process in the blood of the 
human being, which has since been confirmed by others. The further 
development of the fertilized forms, however, does not take place in 
the human blood, but in the bodies of mosquitoes. The fertilized 
organism then penetrates the stomach-wall of the insect, and here 
gives rise to the formation of little cysts, in which after about seven 
days numerous irregular, rounded, ray-like striae appear. After a 
time the capsules of the cysts burst, and the delicate, thread-like 
bodies (the sporozoites) are set free in the body cavity of the mos- 
quito, and shortly after appear in the salivary glands. These bodies 
apparently represent the young parasites, which result from the 
sexual reproduction of the adult organism. If at this stage of 
their development the infested mosquito is allowed to bite a human 
being, malarial infection results, with the appearance in the blood 
of the hyaline forms already described. 



190 THE BLOOD. 

From the above description it will be seen that three forms of 
the malarial parasites may be found in the blood, viz., the parasite of 
tertian, quartan, and sestivo-autumnal fever, and it has been shown 
that these forms may readily be distinguished from each other. 
It should be mentioned, however, that in tertian and quartan fever 
several groups of the same organism may be present at one time, 
and as the process of segmentation coincides with the occurrence of 
a paroxysm, it will readily be seen that the number of paroxysms 
within a given time depends directly upon the number of groups 
which may be present in the blood. If a double infection with the 
tertian parasite has occurred, one group of organisms may thus have 
just reached the segmenting stage, while the second group has at- 
tained only a twenty-four hours' growth, the result being that maturity 
is reached by the two groups on successive days. Quotidian fever 
is then the result. Should still other groups be present, the clinical 
picture will accordingly become more complicated. In quartan 
ague, similarly, double quartan fever will occur if two groups are 
present, and triple quartan fever if three groups are present at one 
time. Mixed infections, further, are also possible. 

In conclusion, it may not be out of place to refer to the presence 
of pigment-bearing leucocytes in the blood of malarial patients. 
These are quite constantly met with during the paroxysm, and it is 
indeed often possible to observe the process of phagocytosis directly 
under the microscope (see Fig. 15). The forms which are taken up 
are the central pigment-clumps of organisms that have undergone 
sporulation, the small, fragmented extracellular forms, the flagellate 
bodies, and even the segmenting bodies. In every case where pig- 
ment-bearing leucocytes — which are probably always of the neu- 
trophilic, polynuclear variety — are observed malarial fever should 
be suspected and a careful examination made, as a melansemia has 
so far been observed only in this disease, in relapsing fever, and in 
connection with the rare melanotic tumors, in which not only leuco- 
cytes containing melanin occur in large numbers, but also masses of 
this pigment float free in the blood. 

Literature. — A. Laveran, Nature parasitaire des accidents de'limpaludisme, 
Description d'un nouveau parasite, Paris, 1881. P. Manson, Tropical Diseases, 
Cassell & Co., London, 1900, p. 1. For a full account of the literature, see the 
monograph by W. S. Thayer and J. Hewetson, " The Malarial Fevers of Baltimore," 
Johns Hopkins Hosp. Eep., vol. v. On recent advances in our knowledge concerning 
the etiology of malarial fever, see W. S. Thayer, Phila. Med. Jour., 1900, p. 1046, 
where a full account of the literature is given. T. B. Futcher, "A Critical Summary 
of Eecent Literature concerning the Mosquito as an Agent in the Transmission of 
Maiaria," Am. Jour. Med. Sci., 1899, p. 318. W. S. MacCallum, "On the Hsematozoon 
Infection of Birds," Jour. Exper. Med., vol. iii. p. 117. E. L. Opie, " On the Haema- 
tozoon of Birds," Ibid., p. 79. F. Grohe, " Zur Gesch. d. Melanaemie," Virchow's Archiv, 
1861, vol. xx. 306. 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 191 

Trypanosomiasis. 

The first authentic report on the occurrence of trypanosomes in 
man was made by Dutton in 1902, while in animals their occasional 
presence had long been known. They have been described in frogs, 
dogs, rats, ground-hogs, and horses, and certain species appear to be 
distinctly pathogenic for some domestic animals in tropical regions. 

Especially interesting is the observation of Castellani and Bruce 
that trypanosomiasis occurs in a large percentage of cases of sleeping 
sickness. Bruce could demonstrate the organism in the blood in 12 
of 13 cases, and in the cerebrospinal fluid obtained by lumbar 
puncture in all of 38 cases. Castellani had previously found the 
same parasite in the cerebrospinal fluid in 20 of 34 cases. The 
question Avhether the disease is caused by trypanosomes is, however, 
not as yet decided. Manson, who likewise found the organism, 
nevertheless expresses himself very reservedly. 



Fig. 35. 




C©*^ 



Trypanosoma gambiense in human blood. (Dutton.) 

The trypanosoma gambiense (Dutton) is from 8 to 25 p. long, and 
from 2 to 2.8 // broad. It is provided with an undulating mem- 
brane and a flagellum, which starts from a centrosome or micronucleus, 
lying in the posterior end of the animal, and projects somewhat 
beyond the anterior end (Fig. 35). There is an oval nucleus which 
is centrally located and is made up of chromatin granules. 

In the wet preparation the organism exhibits slow spiral move- 
ments. It is found free in the blood-plasma, but may also be seen 
in the interior of leucocytes, which latter manifestly destroy the 
organisms exactly as the malarial parasites. In dry specimens the 
trypanosomes can be readily stained with any basic dye ; with the 
RomanoAvsky stain or one of its modifications it is stained like the 
malarial organism. Their number in a blood preparation is rarely 



192 THE BLOOD. 

large ; as a rule not more than from 3 to 8 are found to a cover- 
slip. During apyrexia they are not seen. Infection in man prob- 
ably occurs through mosquitoes. 

Literature. — Dutton, Thompson-Yates Laboratory Eep., 1902, vol. iv. Part II., 
p. 455 ; and Brit. Med. Jour., 1903, vol. i. p. 304. Castellani and Bruce, Ibid., pp. 1218 and 
1431 ; Jour. Trop. Med., 1903, p. 167. 

Spotted Fever. 

In the so-called spotted fever, which occurs in Montana, Nevada, 
Oregon, etc., au intracorpuscular amoeboid, non-pigmented organism 
has been described, which is thought to be the cause of the disease. 
It sometimes has a terminal dark spot and sometimes occurs in pairs, 
when it is not amoeboid. It is termed tne Pyroplasma hominis. 
Infection supposedly takes place through ticks belonging to the 
species Dermaceutor reticulatus. 

Literature. — Wilson and Chowning, Jour. Am. Med. Assoc, 1902, vol. xxxix. p. 
131. Anderson, J. F., Am. Med., 1903, vol. vi. p. 506. 

Filariasis. 

According to Manson, the embryos of at least four, and possibly 
iive and even more distinct species of nematodes may be found in 
the blood of man. These various blood worms Manson designates 
as the Filaria nocturna, Filaria diurna, Filaria perstans, Filaria 
demarquaii, Filaria ozzardi (a doubtful species), and a sixth, which 
may or may not be connected with one of the two last, the Filaria 
magelhsesi. Two of these at least are of pathological import, viz., 
the Filaria nocturna and the Filaria perstans. 

Filaria Nocturna (Manson) : syn. f Filaria sanguinis hominis 
(Lewis). — This filaria is the embryo form of the Filaria Bancrofti 
(Cobbold), which inhabits the lymphatics and is unquestionably the 
cause of endemic chyluria, of various forms of lymphatic varix, of 
tropical elephantiasis arabum, and possibly also of other obscure 
tropical diseases. The organism in question is widely distributed. 
It is indigenous in almost all tropical and subtropical countries as 
far north as Spain in Europe and Charleston in the United States, 
and as far south as Brisbane in Australia. It is very common in 
Cochin and in some of the South Sea Islands, where one-third and 
one-half of the population, respectively, appear to be infested. 

In the following description of both parent and embryo form I 
quote largely from Manson's account of the parasite in his admirable 
manual of tropical diseases. 

The parent filarise are hair-like, transparent worms measuring from 
7.5 to 10 cm. in length. The sexes live together, often inextricably 
coiled about each other. Sometimes they are enclosed, coiled several 
in a bunch, and tightly packed in little cyst-like dilatations of the 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 193 

distal lymphatics ; sometimes they lie more loosely in lymphatic 
varices ; sometimes they inhabit the large lymphatic trunks between 
the glands, the glands themselves, and probably not infrequently the 
thoracic duct. The female is the larger; there are two uterine 
tubes which occupy the greater part of the body, and which are filled 
with ova in various stages of development. The vagina opens near 
the mouth ; the anus just in advance of the tip of the tail. The 
cuticle is smooth and without markings. In both sexes the mouth- 
end tapers slightly ; it is clubbed and simple. The male is charac- 
terized by its marked disposition to curve. The cloaca gives exit to 
two slender unequal spicules. 

In the wet preparations the Filaria nocturna appears as a trans- 
parent colorless little worm, which wriggles about most actively, 
constantly agitating and displacing the corpuscles in its vicinity. It 
will be noticed, however, that the animal does not propel itself 
through the drop of blood, but remains stationary. At first the 



Fig. 36. 




Filaria sanguinis hominis, showing sheath. (After Lewis.) 

movements are so active that it is impossible to make out any ana- 
tomical details ; after a number of hours, however, the movements 
become more sluggish, and it is then possible to study the w r orm 
with more ease. It measures about 0.31 mm. in length by 0.007- 
0.008 mm. in width. With the higher power it will be seen that the 
entire worm is enclosed in a delicate envelope, in which it moves 
backward and forward, the sheath being much larger than the worm 
(Fig. 36). It is owing to the presence of this sheath that active loco- 
motion on the part of the worm is not possible. About the posterior 
part of the middle third of the parasite there is an irregular aggrega- 
tion of granular matter, which represents a viscus of some sort. With 
a high power one can further make out a delicate transverse striation 
in the musculocutaneous layer throughout the entire length of the 
animal. In stained specimens two V-shaped light spots can be 

13 



194 THE BLOOD. 

made out ; one at a point about one-fifth of the entire length of the 
organism, backward from the head-end ; the other very much smaller, 
a short distance from the tail. The first, Manson designates the 
" V " spot, the second the tail spot. In stained specimens these 
two spots are readily made out, as they do not take the color. When 
the movements of the animal have almost ceased, one can see on 
careful focussing that the head is constantly being covered and 
uncovered by a six-lipped or hooked, and very delicate prepuce; 
and, moreover, one can sometimes see a short fang of extreme 
tenuity suddenly shot out from the uncovered extreme cephalic end, 
and as suddenly retracted. 

Technique. — The examination should be made late in the even- 
ing, after the patient has rested for a number of hours. Drops of 
blood are then mounted, wet, on slides and ringed with vaseline to 
prevent the specimen from drying. In such preparations the filarise 
keep alive for a week or longer. They should be searched for with 
a low power — an inch objective is very convenient for the purpose. 
Attention is directed to their presence by the commotion which they 
cause among the neighboring blood corpuscles. 

To prepare permanent mounts, blood-smears are best made on 
slides which are then stained with the eosinate in the usual manner. 
Working with the blood of infected animals, I have thus obtained 
very good results. The V and tail spots are very well brought out. 
To show anatomical details, however, staining with eosin and hsema- 
toxylin after fixing the smears with alcohol or heat, gives the best 
results ; in this manner the sheath is very well shown, as also the 
structure of the musculocutaneous layer. 

The number of worms which may be found in a specimen is very 
variable. During the daytime they are rarely seen, and if at all, 
only one or two specimens at most are found. As evening ap- 
proaches, however, commencing about 5 or 6 o'clock, the filarise 
enter the peripheral circulation in increasing numbers. At mid- 
night the maximum number is about reached, with from 300 to 600 
to the drop of blood. Later they gradually decrease, and by 8 or 
9 A. M. they have again disappeared. This periodicity, however, 
may be reversed if the patient is made to sleep during the daytime 
and remains awake at nights. During their absence from the 
peripheral circulation they may be found in the larger arteries and 
in the lung. 

In non-active cases the number of filarise even at night is quite 
small. In one instance of this kind I found only the sheath of a 
single worm while examining perhaps fifty specimens. 

Infection occurs through the females of certain mosquitoes, prob- 
ably of the genus Culex, which have fed on the blood of a filaria- 
infested individual. The history of the parasite while in the body 
of the mosquito is in brief the following : after their arrival in 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 195 

the stomach the young worms shed the sheath and invade the 
thoracic muscles, where they increase in size (to 1.5 mm.), de- 
velop a mouth, an alimentary canal, and a trilobed tail. They 
then find their way into the abdomen, where, in suitably prepared 
sections, they may occasionally be seen in the tissues about the 
stomach, and even among the eggs in the posterior part of the 
abdomen. The majority now find their way to the base of the pro- 
boscis and under appropriate conditions out through the proboscis 
by a channel which they make for themselves. After introduction 
into the human body the organism finds its way into the lymphatics, 
where it attains sexual maturity ; fecundation takes place and the new 
generation of filarise enter the blood-current by way of the thoracic 
duct and the left subclavian vein. The development of the embryo 
form in the mosquito occupies from sixteen to twenty days. 

Whether or not infection can occur in any other way is not 
known, but not impossible. We could conceive that some of the 
worms are eliminated with the eggs of the mosquito, and that infec- 
tion could then take place through contaminated drinking-water. 

Filaria Perstans. — This species is of interest as it was thought 
to be concerned in the causation of the so-called sleeping sickness of 
west tropical Africa. It has likewise been found in the Buck Indians 
of British Guiana, among whom the same sickness also occurs. 1 The 
organism observes no periodicity, but is present in the blood both 
during the daytime and at night. 

The embryo worm is smaller than the filaria nocturna ; it meas- 
ures about 0.2 mm. in length by 0.004 mm. in breadth. It has 
no sheath, and its caudal end is truncated and abruptly rounded. 
There is no hooked cephalic prepuce. Its motion is progressive. 

The adult form measures 70—80 mm. in length. The tail in both 
sexes is incurvated and the chitinous covering at the extreme tip 
split, as it were, into two minute triangular appendages. They 
have been found in the connective tissue, at the root of the mesen- 
tery, behind the abdominal aorta, and beneath the pericardium. 

Literature. — Mosler u. Peiper, Specielle pathol. u. Therap., 1894, vol. vi. p. 219. 
P. Manson, Allbutt's System of Medicine, vol. ii. I. Guiteras, Med. News, April, 
1886. F. P. Henry, Ibid., 1896. E. Opie. Am, Jour. Med. Sci., 1901, vol. cxxii. p. 251. 
P. Manson, Tropical Diseases, Cassell & Co., London, 1900. 

Distomiasis (Bilharziasis). 

Bilharzia hsematobia (Cobbold) : syn., Gynaecophorus (Diesing) ; 
Distomum haematobium (Bilharz) ; Schistosoma haematobium (Wein- 
land) ; Distoma capense (Harley) ; Thecosoma (Maguin-Tandon). 

The Bilharzia heematobia belongs to the class of trematode 

1 More recent observations tend to throw doubt on this relationship and rather 
suggest a connection between a species of Trypanosoma and sleeping sickness (see 



196 THE BLOOD. 

platodes. According to Bilharz, the greater portion of the Fellah 
and Coptic population of Egypt is infested. It is abundant in 
South Africa, and has also been observed in Mesopotamia, and 
apparently in Arabia. In the United States a few isolated cases 
have been seen which were undoubtedly imported. From Europe 
no endemic cases have been reported. The parasite may give rise 
to diarrhoea, hematuria, and ulceration of the mucous surfaces. 

The male is smaller but thicker than the female, measuring from 
12 to 15 mm. in length by 1 mm. in breadth. On its abdominal 
surface a deep groove is found with overlapping edges, which serves 
for the reception of the female (Fig. 37). It has an oval and a 
ventral sucker placed close together. 

Fig. 37. 




Male and female specimens of the human blood fluke (Bilharzia hxmatobia). X 12. 

(After Looss.) 

The adult parasites are found in the blood of the portal vein, in 
its mesenteric and splenic branches, and in the vesical, uterine, and 
hemorrhoidal veins ; they have also been found in the vena cava 
and may possibly occur elsewhere in the circulation. The eggs 
are more often seen. They are oval bodies, measuring 0.16 
mm. in length by 0.05 mm. in breadth, and are provided with a 
distinct, spike-like projection which issues from one extremity or 
the side (Fig. 38). Infection usually takes place through unfiltered 



BACTERIOLOGY AND PARASITOLOGY OF THE BLOOD. 197 

drinkiDg- water, but may also occur through the skin. Through the 
portal system the parasite then infests the urogenital system, the 

Fig. 38. 





f 





■i 



Bilharzia eggs from the urine: Group a was drawn to scale with B. & L. % obj,, and 1 in. 
ocular ; group b represents their appearance with B. & L. % obj. 

anus, and rectum, and may also proliferate abundantly in the intes- 
tine, the liver, kidneys, etc. The diagnosis is usually made by 
examination of the urine, in which the ova will be found. 

Literature. — Bilharz, Wien. med. Woch., 1856, vol. vi. p. 49. Meissner, 
Schmidt's Jahrbuch., 1882, vol. xx, p. 193. Rutimeyer, Verhandl. d. Cong. f. inn. 
Med., 1822, vol. xi. p. 144. 

Anguilluliasis. 

In 1995 Teissier reported a case of intermittent fever in which 
numerous embryos of anguillula were found in the blood. They 
disappeared after expulsion of the parasites from the intestinal tract, 
and at the same time the fever ceased. It is a question, how- 
ever, whether Teissier's parasite was identical with the common form 
described by Bavay, Normand, Grassi, and others (see page 320). 
Unlike the embryo developing from the eggs of both parasitic and 
free living generations, Teissier's form did not present the charac- 
teristic double oesophageal enlargement, and he reports, moreover, 
that in the case of the adult male only one, instead of two, spicules 
was noted. This view is strengthened by the observation that after 
inoculation into frogs the worms developed in the intestinal canal 
and the lungs into giant forms, which may have been Ascaris nigro- 
venosa (syn., Rhabdonema nigrovenosum). 

Literature.— Teissier, Compt. rend, de Pacad. des sci., 1895, vol. cxxi. p. 171. 
Arch, de med. exper. et d'anat. path., 1895, vol. vii. p. 675; Ibid., 1896, vol. viii. p. 
586. 



CHAPTER II. 

THE SECRETIONS OF THE MOUTH. 

SALIVA. 

Normal saliva is a mixture of the secretions derived from the 
submaxillary, sublingual, parotid, and mucous glands of the mouth. 
It is a colorless, inodorous, tasteless, somewhat stringy and frothy 
liquid, and serves the purpose of aiding in the acts of mastication, 
deglutition, and digestion. The quantity secreted in twenty-four 
hours amounts to about 1500 grammes. 

General Characteristics. 

Normal saliva has a specific gravity of from 1.002 to 1.009, cor- 
responding to the presence of from 4 to 10 grammes of solids. The 
reaction of the saliva proper is alkaline, the degree of alkalinity 
corresponding to from 0.006 to 0.048 per cent, of sodium hydrate. 
Normally an acid saliva is observed only in newly born infants and 
in sucklings. 

The reaction of the tongue and the mucous membrane lining the 
mouth is quite commonly acid early in the morning, owing to the 
production of lactic acid by some of the bacteria which are constantly 
present in the mouth. This acid, according to Magittot, corrodes 
the enamel of the teeth, and may ultimately produce dental caries. 

Chemistry of the Saliva. 

In order to give an idea of the general composition of the saliva 
the following analyses are appended ; the figures correspond to 1000 
parts by weight : 

Water 995.20 994.20 988.10 

Ptyalin 1 1.34 1.30 1.30 

EpThelium} 162 2 ' 20 2 ' 60 

Fatty matter . . 0.50 

Sulphocyanides ........ 0.06 0.04 0.09 

Alkaline chlorides 0.84 . . . . 

Disodium phosphate 0.94 2.20 3.40 

Magnesium and calcium salts . . 0.04 . . . . 

Alkaline carbonates ...... traces. 

1 These figures are too high, as they refer to the total precipitate obtained with 
alcohol. 

198 



SALIVA. 199 

In order to demonstrate the presence of the sulphocyanides, it is 
usually only necessary to heat a few cubic centimeters of the pure 
saliva, faintly acidified with hydrochloric acid, with a dilute solution 
of ferric chloride, when a red color will be seen to develop. If 
necessary, larger quantities, such as 100 c.c, are evaporated to a 
small volume ; the test is then applied to the concentrated fluid. 

Of organic matter, ptyalin, a little albumin mixed with mucin, 
and about 1 gramme of urea pro liter are found. Of all these sub- 
stances, the ptyalin is especially interesting from a physiological 
point of view. It may be isolated in a comparatively pure state 
according to Gautier's method : 

To a large quantity of saliva alcohol (98 per cent.) is added as 
long as a flocculent precipitate forms. This is collected upon a small 
filter and dissolved in a little distilled water. The solution thus 
obtained is treated with several drops of a solution of mercuric 
chloride, in order to remove albuminous material, which is filtered 
off. The excess of mercury is removed by means of hydrogen 
sulphide, when the remaining liquid is evaporated at a temperature 
of from 35° to 40° C, and taken up with strong alcohol. The in- 
soluble residue is dissolved in a little water, filtered, dialyzed in 
order to remove inorganic salts, and is finally precipitated with 
strong alcohol, when the ptyalin will separate out in light flakes. 
Obtained in this manner, ptyalin is a white amorphous substance, 
soluble in water, dilute alcohol, and glycerin. In neutral or even 
slightly alkaline solutions, but not in acid solutions, it rapidly 
transforms boiled starch into dextrin and sugar at a temperature 
of from 35° to 40° C This transformation takes place according 
to the equations : 

(1) (C 12 H 20 O 10 ) 54 -f 3H 2 = 3[(C, 2 H 20 O 10 ) 17 .C, 2 H 22 O 1 ,]. 

Starch. Erythrodextrin. 

(2) 3[(C 12 H 20 O 10 ) n .C 12 H 22 O n ] + 6H 2 = 9[(C 12 H 20 O 10 ) 5 .C 12 H 22 O u ]. 

Erythrodextrin. Achroodextrin 

(3) 9[(C 12 H 20 O 10 ) 6 .C 12 H 22 O n ] + 45H 2 = 54C 12 H 22 O n = 54C 12 H 22 O n . 

Achroodextrin. Isomaltose. Maltose. 

In order to test for ptyalin, a few cubic centimeters of saliva are 
filtered and added to a solution of starch ; the mixture is placed in 
the warm chamber for some time, when it is tested with cupric sul- 
phate or iodine. At first, starch gives a blue color with iodine ; 
after the reaction has proceeded further a red or violet-red color is 
obtained, indicating the presence of erythrodextrin, while no change 
in color at all results when achroodextrin only is present. The 
maltose may be recognized by the fact that it turns the plane of 
polarization more strongly to the right than glucose ; it also reduces 
Fehling's solution. 

The test for nitrites, which may likewise be present in the saliva, 
is conducted in the following manner : about 10 c.c. of saliva are 



200 



THE SECRETIONS OF THE MOUTH. 



treated with a few drops of Ilasvay's reagent and heated to a tempera- 
ture of 80° C, when in the presence of nitrites a red color will 
develop. The reagent is prepared as follows : 0.5 gramme of sulph- 
anilic acid in 150 c.c. of dilute acetic acid is treated with 0.1 
gramme of naphtylamin dissolved in 20 c.c. of boiling water. After 
standing for some time the supernatant fluid is poured off and the 
blue sediment dissolved in 150 c.c. of dilute acetic acid. The solu- 
tion is kept in a sealed bottle. 



Microscopical Examination of the Saliva. 

If normal saliva is allowed to stand, two layers will be seen to 
form, viz., an upper clear and a lower cloudy layer, which latter con- 
tains certain morphological elements. Among these, salivary cor- 
puscles, pavement epithelial cells, and micro-organisms are found 
(Fig. 39). 

Fig. "39. 




Buccal secretion. (Eye-piece III., obj. Reichert, ^ homogeneous immersion : Abbe's 
mirror, open condensers.) a, epithelial cells ; b, salivary corpuscles ; c, fat-drops ; d, leuco- 
cytes ; e, Spirochaeta buccalis ; /, comma-bacillus of mouth; g, Leptothrix buccalis; h, i, k, 
various fungi, (v. Jaksch.) 

The salivary corpuscles resemble white corpuscles very closely, 
but differ in their greater size and coarser appearance. The epi- 
thelial cells are large, irregular, polygonal cells, provided with well- 
defined nuclei and nucleoli ; they exhibit certain irregularities in 
size, according to their origin, and belong to the class of pavement 
or stratified epithelium. 

Micro-organisms. 1 — While schizomycetes and moulds are only 
exceptionally found in the mouth under normal conditions, and are 
then undoubtedly derived from ingested food, bacteria are always 
present in large numbers, and it is not surprising that all forms 
which are found in the air, food, and drink may here be encountered. 
Some of these, such as the Leptothrix buccalis innominata, Bacillus 
buccalis maximns, Leptothrix buccalis maxima, Iodococcus vagi- 

1 W. D. Miller, Die Mikroorganismen d. Mundhohle, 1892. 



SALIVA. 201 

natus, Spirillum sputigenum, and Spirochete den ti urn, are always 
present. Together with other bacteria, they have been found in 
carious teeth, in abscesses communicating with the mouth and 
pharynx, and in exudates on the mucous membranes of these parts. 
In all probability, however, they are non-pathogenic. To this class 
also belongs the smegma bacillus, which has been encountered in the 
saliva, the coating of the tongue, and in the tartar of the teeth of 
perfectly healthy individuals. In this connection it is interesting to 
note that, in contradistinction to the bacteria which are only tem- 
porarily found in the mouth, the majority of those which are con- 
stantly present cannot be cultivated on artificial media. 

Important from a practical standpoint is the fact that a number 
of pathogenic micro-organisms may at times be found under normal 
conditions. The Diplococcus pneumoniae, also known as the pneu- 
mococcus of Frankel and Weichselbaum, the Diplococcus lanceolatus, 
the Micrococcus lanceolatus, the Micrococcus septicaemia? sputi, and 
the Micrococcus pneumoniae cruposse (Sternberg), has thus been found 
in a virulent condition in from 15 to 20 per cent, of healthy indi- 
viduals, and it is even claimed that in a non-virulent state it is 
constantly present in the mouth. Streptococci are likewise frequently 
observed, but usually possess but little virulence or none at all 
when obtained from the healthy mouth and tested upon animals. 
Pyogenic staphylococci may also be found at times, but are less 
common than the streptococci. Most important is the occasional 
occurrence of the diphtheria bacillus in the mouths of individuals 
who have not been exposed to contagion. Welch x mentions that 
virulent organisms were found by Park and Beebe in the healthy 
throats of eight out of three hundred and thirty persons in New 
York, who gave no history of direct contact with cases of diphtheria. 
Two of these eight persons later developed the disease. Non-virulent 
bacilli were found in twenty-four individuals of the same series, and 
the pseudodiphtheria bacillus in twenty-seven. 

Other pathogenic bacteria which may be found in normal mouths 
are the Micrococcus tetragenus, the Bacillus pneumoniae of Fried- 
lander, the Bacillus crassus sputigenus, and the Bacillus coli com- 
munis. 

It is interesting to note that the secretions of the mouth and 
throat, as most secretions of the body, possess a certain degree of 
germicidal power. The Staphylococcus aureus, the Streptococcus 
pyogenes, the Micrococcus tetragenus, the typhoid bacillus, and the 
cholera spirillum, when present in moderate numbers, are thus 
killed by the saliva. The diphtheria bacillus, however, is more 
resistant, and may survive for twenty-four to forty days. It has 
been found, as a matter of fact, that the organism may be demon- 
strated in the throats of some individuals who have passed through 

1 Dennis' System of Surgery : Surgical Bacteriology. 



202 THE SECRETIONS OF THE MOUTH. 

an attack of diphtheria for several weeks after all the clinical symp- 
toms have disappeared. The Diplococcus pneumoniae is even said 
to grow well in saliva, although it rapidly loses its virulence. By 
then cultivating it upon pneumonic sputum, however, the virulence 
of the organism is restored. The individual bacteria will be con- 
sidered in detail later on. 



Pathological Alterations. 

It has been mentioned that about 1500 grammes of saliva are 
secreted in the twenty-four hours. This quantity is, however, sub- 
ject to great variation. An increase is thus frequently noted in 
pregnancy, in various neurotic conditions, in tabes, bulbar paralysis, 
in inflammatory diseases of the mouth, in dental caries, following 
the administration of pilocarpin, in poisoning with mercury, acids, 
and alkalies, etc. The quantity is diminished in all febrile diseases, 
in diabetes, and often in nephritis. The effect of psychic influences 
upon the secretion of saliva as well as of other glands is well 
known, an increase or decrease in the flow being produced under 
various conditions. 

In determining whether or not salivation actually exists, the physi- 
cian should not only be guided by the statements of his patients, 
but an actual estimation of the amount secreted within a definite 
period of time should be made. Hysterical individuals not infre- 
quently complain of "salivation," when a direct estimation will 
show that the amount is not only not increased, but actually dimin- 
ished. 

An acid reaction of the saliva has been noted in various diseases 
of the intestinal tract, in febrile diseases, and notably in diabetes 
(Frerichs). According to Strauss and Cohn, however, an alkaline 
reaction of the saliva is the rule even under pathological conditions. 

Among the qualitative changes may be mentioned an increase iu 
the amount of urea, which has been repeatedly observed, and especi- 
ally in nephritis. 

Urea may be demonstrated as follows : the saliva is extracted 
with alcohol, the nitrate evaporated, and the residue dissolved in 
amyl alcohol. This is allowed to evaporate spontaneously, when 
crystals of urea will separate out, and may then be examined micro- 
scopically and chemically (see Urine). 

Bile-pigment and sugar have not been found in the saliva. 

Of drugs, potassium iodide and potassium bromide rapidly pass 
into the saliva. Upon this property the indirect examination of the 
gastric juice for its digestive power — i. e., the presence or absence 
of free hydrochloric acid — by means of the potassium iodide and 
fibrin packages of Giinzburg, is partly based. 

In order to test for potassium iodide, strips of filter-paper moist- 



SPECIAL DISEASES OF THE MOUTH. 203 

ened with starch solution are immersed in the saliva, which has 
been acidified with nitric acid ; in the presence of potassium iodide 
the starch-paper turns blue. 

SPECIAL DISEASES OF THE MOUTH. 

Tuberculosis. — In cases of lupus and the so-called benign form 
of tuberculosis of the mouth it is rarely possible to demonstrate the 
presence of tubercle bacilli, even in scrapings taken from the base 
of the ulcers or in the diseased tissue itself, while in cases of ulcer- 
ative stomatitis associated with phthisis in its advanced stages they 
may be frequently found in large numbers. In some cases, however, 
their demonstration is by no means easy. In the saliva they are 
only exceptionally seen. 

Actinomycosis. — In cases of actinomycosis it is occasionally pos- 
sible to demonstrate the presence of the specific organism in or about 
carious teeth. More commonly, however, the patients are not seen 
until the primary symptoms of the disease have disappeared, when 
the typical kernels can no longer be found at the original points 
of entry or have become unrecognizable owing to calcification and 
retrogressive changes. 

Usually the disease has already progressed to the formation of a 
distinct tumor or abscess, and it may then be necessary to make an 
exploratory incision, and to examine the scrapings which are brought 
away. The number of kernels which may be found is at times very 
small, but a careful examination will probably always lead to their 
detection if the disease in question is actinomycosis. 

Catarrhal Stomatitis. — In this affection the quantity of saliva 
is increased. Microscopically an increased number of epithelial 
cells and many leucocytes are noted, their number depending upon 
the intensity of the morbid process. 

Ulcerative Stomatitis. — In this condition, following mercurial 
poisoning or scurvy, the same appearance is noted microscopically 
as in simple stomatitis. In addition there may be necrotic tissue, 
red blood-corpuscles, and innumerable leucocytes. The reaction of 
the saliva is intensely alkaline, the color markedly brown, and its 
odor fetid. 

Gonorrhoeal Stomatitis. — The number of cases of gonorrhoeal 
stomatitis that have thus far been recorded is small. The disease, 
however, has received but little attention, and is probably more 
common than is generally supposed. In the adult it may be con- 
tracted through coitus contra naturam, while in the newborn the 
infection is undoubtedly brought about in the same manner as the 
corresponding disease of the conjunctiva. In suspected cases the 
exudate which forms upon the gums, the tongue, and the palate 
should be examined for the presence of gonococci. In adults the 



204 



THE SECRETIONS OF THE MOUTH. 



organism has thus far not always been found ; in the newborn, 
however, Rosinski has succeeded in demonstrating its presence in 
all cases examined. 

Thrush. — Oidium albicans (Fig. 40) is most commonly seen in 
children, but may also occur in adults, and especially in phthisical 
individuals, and sometimes lines the entire mouth. If in such cases 
a bit of the membrane is pulled off and examined microscopically, it 
will be found to consist of epithelial cells, leucocytes, and granular 
detritus, with a network of branching, band-like formations, which 



Fig. 40. 




Oidium albicans, the vegetable parasite of muguet or thrush. (Reduced from Ch. Robin.) 

present distinct segments. The contents of the segments are clear, 
and usually contain two highly refractive granules — the spores, one 
of which is situated at each pole. These segments diminish in size 
toward the end of each band, their contents at the same time becom- 
ing slightly granular. 

TARTAR. 

In a bit of tartar scraped from the teeth actively moving spiro- 
chetal are seen, as well as long, usually segmented bacilli, frequently 
forming bands which are colored bluish red by a solution of iodo- 
potassic iodide. Leptothrix buccalis, shorter bacilli (which are not 
colored by this reagent), micrococci, and a large number of leuco- 
cytes and epithelial cells which have undergone fatty degeneration, 
are also found. Infusoria have been found by Sternberg, P. Cohn- 
heim, v. Leyden, and others, v. Leyden states that he found infu- 
soria in the tartar in his own person. 

COATING OF THE TONGUE. 

A brown coating of the tongue is often observed in severe infec- 
tious diseases, and consists of remnants of food and incrustated blood. 
Microscopically, in addition to a large number of epithelial cells, 
enormous numbers of micro-organisms and a large number of dark, 



PLATE XIII. 



FIG. 1. 



mM 




mim 



Bacteria of the Mouth. (Cornil Babes.) 



FIG. 2. 




Leptotririx Biacealis. (v. Jakseh.] 



COATING OF THE TONSILS. 205 

cell-like structures, probably derived from desquamated epithelial 
cells, are found. The white coating of the tongue contains epithelial 
cells, many micro-organisms, and a few salivary corpuscles. 



COATING OF THE TONSILS. 

Pharyngomycosis Leptothrica. 

In the pyoid masses derived from the crypts of the tonsils in cases 
of follicular tonsillitis, and also in persons who have had frequent 
attacks of tonsillitis, large numbers of lymphocytes of all sizes are 
seen besides epithelial cells and long, segmented fungi — the Lepto- 
thrix buccalis (Plate XIII.) — which are colored bluish red by a solu- 
tion of iodo-potassic iodide. Ordinary polynuclear neutrophils are 
only present in small numbers. At times patches composed of these 
fungi extend over a considerable area of the tonsils, so that it may 
be doubtful whether or not the disease is a beginning diphtheria. A 
microscopical examination will in such cases settle all doubt. 

Tonsillitis. 

In tonsillitis a large number of bacteria have been isolated from 
the pseudomembranous deposits. Among the more important which 
are supposed to bear a causative relation to the disease may be 
mentioned the various streptococci, staphylococci, less commonly the 
pneumococcus, the diplococcus of Brison, the bacillus coli communis, 
the bacillus of Friedlander, the bacillus septicsemiaB sputi, and in a 
few isolated instances the micrococcus tetragenus. In many cases 
in which tonsillar deposits are clinically regarded as diphtheritic 
culture reveals only an abundance of the thrush fungus. 

Meyer, 1 in v. Leyden's clinic, succeeded in cultivating a cliplo- 
streptococcus from the tonsils of five, cases of acute rheumatism with 
angina, and reports that bouillon cultures of the organism produced 
characteristic polyarticular arthritis in rabbits. The same organism 
apparently was also obtained by Allaria 2 in Bozzolo's clinic, and it 
is interesting to note that his cases resulted from manifest contagion. 

In certain cases of ulcerative angina a fusiform diplobacillus and 
spirochaetaB have been found in the exudate by Vincent, 3 Bernheim, 
and others. The only cases reported from the United States thus 
far are those of Mayer 4 and Fisher. 5 

*F. Meyer, Deutsch. rned. Woch., 1901, vol. xxvii. p. 81. 

2 Allaria, Kevista critica di clinica Medica, 1901, vol. ii. p. 805. 

3 Vincent, Bull, de la soc. de Hop., March 11, 1898. 

4 Mayer, Am. Jour. Med. Sci., 1902, vol. cxxiii. p. 187. 
5 Fisher, Ibid., 1903, vol. cxxvi. p. 438. 



206 THE SECRETIONS OF THE 3WUTH. 

Glandular Fever. 

According to Neumann and Comby, glandular fever generally 
depends upon infection with a streptococcus. In the case reported 
by Lande and Froin and by Hirtz 1 bacteriological examination of 
the throat at the height of the febrile stage revealed the presence of 
the pneumococcus in a virulent condition. 



Diphtheria. 

Recognizing the great importance of an early diagnosis in such a 
malignant disease as diphtheria, an examination for Loffler's bacillus 
has become just as important to-day as that for the bacillus of 
tuberculosis. 

By means of a sterilized stout platinum loop, a pair of forceps, or 
a cotton swab, a piece of membrane is scraped from the tonsils, the 
soft palate, or the pharynx, and is at once transferred to a sterilized 
test-tube closed with a pledget of cotton. A thin smear is then 
made either on cover-glasses or slides. If no membrane can be 
procured, smears are made from a cotton mop. When dry, the 
specimens are fixed by being passed three or four times through the 
flame of a Bunsen burner, when they are ready for staining. For 
this purpose, Loffler's alkaline solution of methylene-blue, which 
consists of 30 c.c. of a concentrated alcoholic solution of methylene- 
blue in 100 c.c. of an aqueous solution of potassium hydrate 
(1 : 10,000) may be advantageously employed, the specimen being 
stained for from five to ten minutes. It is then rinsed in water, 
placed on a slide, the excess of water removed with filter-paper, and 
examined with a yL- oil-immersion lens. 

A rapid method of staining, and one which gives even more satis- 
factory results than that of Loffler, is suggested by Neisser. The 
organism is grown on blood-serum and examined after from nine to 
twenty-four hours. The air-dried smears are placed for from one to 
three seconds in a solution composed of 20 c.c. of an alcoholic solu- 
tion of methylene-blue (1 to 20 c.c. of 90 per cent, alcohol), 950 c.c. 
of distilled water, and 30 c.c. of glacial acetic acid. They are then 
washed in water, stained for from three to five seconds in an 0.2 per 
cent, hot and filtered aqueous solution of vesuvin, again washed off, 
dried in the air, and mounted in balsam. The bacilli are brown and 
have in their interior 2 to 4 blue granules which are usually located 
near the poles. 

A dahlia -methyl-green solution may likewise be employed. This 
consists of 10 grammes of a 1 per cent, aqueous solution of dahlia- 
violet and 30 grammes of a 1 per cent, aqueous solution of methyl- 
green. The specimen is stained for from one to two minutes. 

1 Lande et Froin, Eev. mensuelle des Mai. del'Enfance, 1901, p. 78. 



COATING OF THE TONSILS. 207 

The following method also may be employed, as suggested by 
Schauffler. The staining reagent has the following composition : 

Filtered solution of Loffler's methylene-blue 10.0 c.c. 

Filtered solution of pyronin (0.5 gramme to 10 c.c. of water) . . 1.5 c.c. 
Acid alcohol (3 c.c. of 25 per cent, hydrochloric acid to 97 c.c. 

of absolute alcohol) 0.5 c.c. 

Cover-glass specimens are stained for one minute ; they are then 
washed in running water and mounted in balsam as usual. The 
bacilli are stained blue, the pole bodies a bright ruby red. 

Pseudodiphtheritic bacilli are said to take only the blue stain with 
this method. 

If it is desired to employ Gram's method, the specimens are most 
conveniently stained for three minutes with a freshly prepared con- 
centrated alcoholic solution of gentian-anilin water. This is made 
by adding anilin oil to 10 c.c. of distilled water, drop by drop, 
thoroughly shaking after the addition of each drop, until the solution 
becomes opaque. It is then filtered and treated with 10 c.c. of 
absolute alcohol and 11 c.c. of a concentrated alcoholic solution of 
gentian-violet, methyl-violet, or Victoria-blue. The specimen is 
decolorized in a solution composed of 1 gramme of iodine and 2 
grammes of potassium iodide in 300 c.c. of water. After remaining 
in this solution for five minutes the specimen is differentiated in 
95 per cent, alcohol until it ceases to lose color. It is transferred 
to absolute alcohol, then to oil of cloves or xylol, and mounted in 
balsam. If desired, one can give the specimen a counterstain, before 
clearing it, with Bismark-brown. The bacilli retain the blue color. 

Cultures should be made, preferably on a mixture of blood-serum 
and bouillon, as recommended by Loftier. This is composed of 3 
parts of blood-serum and 1 part of bouillon, containing 10 per 
cent, of peptone, 3 per cent, of grape-sugar, and 0.5 per cent, of 
sodium chloride, the mixture being solidified in the usual manner. 
Upon this medium Loffler's bacillus grows so much more rapidly 
than other organisms which are usually present in the secretions of 
the mouth and throat, that, after from six to eight hours' incubation 
at 34° to 35° C, they often form the only colonies that attract 
attention. Smears are then made and stained according to Neisser's 
method. 

In the absence of blood-serum bouillon, alkaline boullion, nutrient 
gelatin, nutrient agar, glycerin-agar, and potato may be employed. 
Coagulated egg-albumin, as pointed out by Booker, and milk are 
also good media. But it is to be noted that the " typical " staining 
effect with Neisser's method is commonly only obtained if the 
organism has been grown on ox-blood serum, and if the growth is 
not older than twenty-four hours. 

The colonies are large, round, elevated, and grayish white in 



208 



THE SECRETIONS OF THE MOUTH 



color, with a centre that is more opaque than the slightly irregular 
periphery. The surface of the colony is at first moist, but after a 
day or two it assumes a dry appearance. 

The bacillus (Fig. 41) is non-motile and varies in size and shape, 
its average length being from 2.5 to 3/z, its breadth from 0.5 to 
0.8 a. Its morphological characteristics are so peculiar as to render 
its identification upon cover-slip preparations and in sections of the 
diphtheritic membrane an easy matter in most cases. 



Fig. 41. 




Bacillus diphtherias : A, its morphology on glycerin-agar-agar ; B, its morphology on Loffler's 
blood-serum ; C, its morphology on acid blood-serum mixture. (Abbott.) 

Sometimes the organism appears as a straight or slightly curved 
rod ; but especially characteristic are irregular and often bizarre 
forms, such as rods with one or both ends terminating in little bulbs 
and rods apparently broken at intervals, in which short, well- 
defined round, oval, or straight segments can be made out. Very 
commonly two organisms lie together forming an obtuse angle, or 
numbers of them may be observed lying side by side. 

Some forms stain uniformly, others in an irregular manner ; the 
most typical appearance, as I have already stated, is that of little 
granules near the poles of the bacillus, which stain blue with 
Neisser's method, while the body of the organism is colored brown. 

Streptococci are also seen, as a rule, and it may be said that the 
gravity of a case is directly proportionate to the number of strep- 
tococci present. 

It is important to note that diphtheria bacilli may still be found 
in the throat for weeks after all clinical symptoms have disappeared. 



COATING OF THE TONSILS. 209 

Patients should hence be isolated until a bacteriological examination 
has demonstrated the absence of the organism. 

Literature. — S. Flexner, " The Bacteriology and Pathology of Diphtheria," 
Johns Hopkins Hosp. Bull., 1895, p. 39. W. H. Welch, Am. Jour. Med. Sci., 1894. 
Heubner, Schmidt's Jahrbucher d. gesammten Med., 1892, vol. ccxxxv-i. p. 270. 
Klebs, Arch. f. exper. Path., 1875, vol. iv. p. 207. Lomer, Centralbl. f. Bakt. u. 
Parasit., 1887, vol. ii. p. 105; and 1890, vol. vii. p. 528. C. Frankel, " Die Unter- 
scheidung d. echten u. d. falschen Diphtheriebacillen," Berlin, klin. Woch., 1897, p. 
1087. W. G. Schauffler, Med. Eecord, Dec. 6, 1902. 

Scarlatina. 

According to Baginsky, streptococci are practically constantly 
found in the pharyngeal secretion. 

Literature.— A. Baginsky, Deutsch. med. Woch., Oct. 23, 1902. 
14 



CHAPTEE III. 
THE GASTRIC JUICE AND GASTRIC CONTENTS. 

THE SECRETION OF GASTRIC JUICE. 

The gastric juice is the result of the glandular activity of the 
stomach, and is the only secretion of the digestive tract which pre- 
sents an acid reaction. 

As is well known, the mucous membrane of the stomach is cov- 
ered throughout its entire extent by a single layer of cylindrical 
epithelium, which dips down in places to line the orifices and larger 
ducts of the numerous tubular glands with which it is beset. Of 
these, two kinds are described, viz., the fundus and pyloric glands, 
so named from the location in which they are principally found. 
In the secretory portion of a fundus gland two sets of cells can be 
distinguished. The one kind is small, granular, and polyhedral or 
columnar, bordering upon the narrow lumen of the tube ; these are 
termed the chief or principal cells (Heidenhain), but are also known 
as the central or adelomorphous cells. They stain with anilin dyes 
to only a slight extent. The others, known as parietal, adelomor- 
phous, or oxyntic cells, are variously situated between the adelomor- 
phous cells and the membrana propria ; they are most numerous 
in the necks of the glands. They are larger than the chief cells, oval 
or angular and finely granular in structure ; they possess a strong 
affinity for the anilin dyes. The pyloric glands, which are found 
only in the region of the pylorus, on the other hand, are character- 
ized by the greater length of their ducts, which are also lined by 
the cylindrical epithelium of the mucous membrane proper. The 
secretory portion of these glands is represented by a single layer of 
short and finely granular, columnar cells, which closely resemble the 
chief cells of the fundus glands. In addition to these, a few isolated 
cells, the cells of Nussbaum, are found, which in structure and in 
their behavior to anilin dyes resemble the parietal cells. 

Upon chemical examination the gastric juice is found to consist 
essentially of water, free hydrochloric acid, pepsin, rennet (a milk- 
curdling ferment), mucus, and certain mineral salts. 

Of these constituents, the hydrochloric acid is secreted by the 
parietal cells, pepsin and the milk-curdling ferment by the chief 
cells of the fundus and the pyloric glands, while the mucus is the 
product of the cylindrical goblet-cells lining the stomach and the 

210 



TEST-MEALS. 211 

wider portions of its glandular ducts. It should be borne in mind, 
however, that the ferments mentioned do not exist in the cells as 
such, but as zymogens, which are transformed into the ferments 
through the activity of the free hydrochloric acid. According to 
modern investigations, moreover, the zymogens only are secreted by 
the cells. 

Until recently it was supposed that the gastric juice is secreted only 
upon appropriate stimulation of the nervous mechanism of the stom- 
ach, either directly or indirectly, and that the stomach in its quiescent 
state — i. <?., when not digesting — is empty. The researches of 
Schreiber' and Martius, however, have rendered the correctness of 
this view doubtful, as they were able to obtain quantities of gas- 
tric juice, varying from 1 to 60 c.c, from the non-digesting stom- 
ach of every normal person examined. , I have likewise never failed 
to obtain a few cubic centimeters under the same conditions. 



TEST-MEALS, 

Although the secretion of gastric juice takes place continuously, 
the amount that can usually be obtained from the non-digesting 
organ is not sufficient for analytical purposes. It is, therefore, nec- 
essary to stimulate the glandular apparatus of the stomach to in- 
creased activity. This may be accomplished with thermic, chemical, 
electrical, and digestive stimuli, of which the last named are the 
most convenient and the most effective, furnishing an idea not only 
of the secretory, but also of the motor and resorptive activity of the 
organ. The analytical results will, however, depend to a large ex- 
tent upon the character of the food ingested, starches and fats exert- 
ing but a slight stimulating effect, while proteids cause a copious 
secretion of gastric juice. The ingestion of fluids at the same time 
will likewise influence the results obtained, owing to dilution of 
the gastric juice. The time of the height of digestion, moreover, 
varies with the kind and quantity of food taken. In order to obtain 
uniform results it is necessary, therefore, to withdraw the gastric 
contents at a certain period after the ingestion of a meal of known 
composition and bulk. 

Numerous test-meals have been proposed. The following are the 
most important : 

The Test-breakfast of Ewald and Boas. 

This consists of from 35 to 70 grammes of wheat-bread and of 
300 to 400 c.c. of water or weak tea, without sugar. It is best to 
give this meal to the patient early in the morning, when the stomach 
is empty — i. e., as a breakfast. The gastric contents are obtained 
one hour later. 



212 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

The Test-dinner of Riegel. 

This consists of a plate of soup (400 c.c.), a beefsteak (200 
grammes), a slice or two of wheat-bread (50 grammes), and a glass- 
ful of water (200 c.c). The contents of the stomach are obtained 
after four hours. The disadvantage of this method lies in the fact 
that the lumen of the stomach-tube is frequently occluded by large 
pieces of undigested meat, a source of annoyance which may be 
guarded against, however, by using finely chopped meat. 

The Double Test-meal of Salzer. 

For breakfast the patient receives 30 grammes of lean, cold roast, 
hashed or cut into strips sufficiently small not to obstruct the 
stomach- tube ; 250 c.c. of milk ; 60 grammes of rice ; and one soft- 
boiled egg. Exactly four hours later the second meal is taken, con- 
sisting of 35 to 70 grammes of stale wheat-bread and 300 to 400 
c.c. of water. The gastric contents are withdrawn one hour later. 
In this manner the gastric juice is not only obtained at the height of 
digestion, but an idea may at the same time be formed of the motor 
power of the stomach. Under normal conditions the organ should 
contain no remnants of the first meal at the time of examination. 

The Test-breakfast of Boas. 

This consists of a plateful of oatmeal-soup, prepared by boiling 
down to 500 c.c. one liter of water to which one tablespoonful of 
rolled oats has been added. A little salt may be used if desired, 
but nothing more. The contents of the stomach are obtained one 
hour later. This test-meal was devised by Boas in order to guard 
against the introduction from without of lactic acid, which is present 
in all kinds of bread. The meal is employed in cases of suspected 
cancer of the stomach in which a quantitative estimation of lactic 
acid is to be made, the stomach being washed out completely the 
night before. 

Still other test-meals have been suggested, but they possess no 
material advantage over those described. 

THE STOMACH-TUBE. 

The stomach-tubes in general use are essentially large Nelaton 
catheters. They should measure at least from 72 to 75 cm. in 
length, and be provided with three fenestra, of which one is placed 
at the end of the tube and two laterally, as near the end as possible. 
For the purpose of washing out the stomach the tube is connected 
with a glass funnel by means of ordinary rubber tubing, which can 
be detached from the stomach-tube proper. There is no advantage 
in rubber funnels or in having a continuous tube. 



THE STOMACH-TUBE. 



213 



It is important that the tubes should be thoroughly cleansed in 
hot water as soon after use as possible. The advice of Boas, more- 
over, to have special, marked tubes for tubercular, syphilitic, and 
carcinomatous patients, should be borne in mind. Patients in whom 
lavage is to be practised for any length of time should provide their 
own instruments. 



Contraindications to the Use of the Tube. 

Of direct contraindications to the use of the tube, there should 
be mentioned the existence of the various forms of valvular disease 
when in a state of imperfect compensation, angina pectoris, arterio- 
sclerosis of high degree, aneurism of the large arteries, recent hem- 
orrhages from whatever cause, marked emphysema with intense 
bronchitis, acute febrile diseases, etc. 

Introduction of the Tube. 



Fig. 42. 



The technique of the introduction of the 
tube should be as simple as possible ; the 
exhibition of complicated bottle arrange- 
ments for the purpose of obtaining the 
gastric juice only adds to the excitement of 
a nervous patient, and should be avoided. 
The patient's clothing and floor of the room 
should be protected from being soiled by 
material that may be vomited along the 
sides of the tube, the dribbling of saliva, 
etc. For this purpose, Tiirck's rubber bib 
with pouch may be advantageously employed. 
" It is so arranged as to form a pouch in 
front, to catch the saliva or stomach contents 
that may be thrown off from the mouth or 
stomach. A detachable tube passes from 
the bottom of the pouch and is conducted 
into a basin or any suitable vessel." 1 

Cocainization of the pharynx is not nec- 
essary, but may be resorted to in hyperges- 
thetic individuals, a 10 per cent, solution 
being employed. 

The tube, held like a pen, is passed to the posterior wall of the 
pharynx, the patient bending his head forward, and not backward, 
as is usually advised. The patient is then told to swallow, but this 
is not^ necessary. The tube is pushed on until resistance is felt 
when it meets with the floor of the stomach. The procedure does 
not occupy ten seconds. At the least sign of cyanosis or of marked 

1 Manufactured by G. Tiemaun & Co., New York. 




Boas' bulbed tube. 



214 



THE GASTRIC JUICE AND GASTRIC CONTENTS. 



pallor the tube should be withdrawn at once, and the patient ob- 
served for a day or two before a second attempt is made. 

If the gastric juice does not flow at once, the patient is instructed 
to bear down with his abdominal muscles, and, if this is insufficient, 
to cough a little. Repeated attempts of this kind will usually bring 
about the desired result, unless the tube has not been introduced far 
enough or too far ; in the latter case it will double upon itself, so 
that its end rises above the level of the liquid. Pressing upon 
the abdomen with the hands is of no effect (Method of Expression). 

Aspiration must at times be employed. For this purpose', Boas' 
bulbed tube (Fig. 42) is convenient. The manner in which it is 
used is the following i the proximal end of the tube, after having 
been introduced into the stomach, is compressed and the bulb 
squeezed, when the distal end is clamped and the bulb allowed to 
expand. When this is repeated several times a partial vacuum is 



Fig. 43. 




Arrangement of bottle for aspiration of the gastric contents. 

produced in the tube, which usually causes a flow of gastric juice. 
In the absence of such an instrument the stomach-tube may be con- 
nected with a bottle, in which a partial vacuum has been established 
by aspiration (Fig. 43). Unless the patient is accustomed to the 
introduction of the tube, however, these more complicated proced- 
ures should be avoided as much as possible (Method of Aspiration). 
I have found that in cases in which gastric juice cannot be ob- 
tained by expression the flow may often be started by suction with 
the mouth, and I regard this method as preferable to the one just 
described. With due precautions, viz., holding the tube between the 
fingers near the mouth of the patient, so as to be informed at once, 
by the sense of touch, when the stomach contents have reached this 
point, unpleasant results will be obviated. If only a very small 
amount of gastric juice is present in the stomach — i. e., when a defi- 



GENERAL CHARACTERISTICS OF THE GASTRIC JUICE. 215 

nite flow cannot be established — it is best to suck lightly with the 
mouth, to compress the tube firmly, to remove it as rapidly as pos- 
sible, and empty it into a little dish. A few drops, sufficient to test 
for free hydrochloric acid, can thus always be obtained, even from 
the non-digesting organ. 

Mnhorn's bucket-method is of little value, as the amount of gastric 
juice which can thus be obtained is insufficient for analytical pur- 
poses. It may be employed, however, in patients who are particu- 
larly nervous, and who object to the use of the tube, and possibly 
also when its use is contraindicated. The test for hydrochloric 
acid can be made, but the information thereby obtained is in itself of 
comparatively little value. 

In order to wash out the stomach, the funnel-tube is attached, the 
funnel filled with lukewarm water or any desired medicated solution, 
elevated above the head of the patient, and the water allowed to 
flow. From 500 to 1000 c.c. may be introduced at one time. By 
suddenly depressing and inverting the funnel over a suitable vessel 
before all water has left the funnel a siphon arrangement is estab- 
lished and the stomach emptied. It is well to measure the return- 
ing water as well as the amount introduced. Should the flow 
diminish or cease before all the water has been removed, the end 
of the tube probably stands above the level of the liquid, and the 
flow can be started again by pushing the tube on further or by 
withdrawing it a little, as the case may be. 

Washing out the stomach soon after the ingestion of a full meal is 
always very tedious and annoying, if not an impossible procedure, 
as the fenestra readily become obstructed. Should this occur, the 
funnel, filled with water, is elevated as high as possible, with a view 
to overcome the obstruction by hydrostatic pressure ; or, if this 
proves insufficient, the funnel-tube is detached and the obstruction 
dislodged by means of air, for which purpose a Politzer bag or the 
bulb of a Boas tube is very convenient. 

GENERAL CHARACTERISTICS OF THE GASTRIC JUICE. 

Pure gastric juice is an almost clear, faintly yellowish fluid, of a 
sour taste and a peculiar, characteristic odor. Its specific gravity 
varies between 1.002 and 1.003, corresponding to the presence of 
bat 0.5 per cent, of solids. Its reaction, owing to the presence of 
hydrochloric acid, is acid. 

Amount. 

Very little is known of the total quantity of gastric juice that is 
secreted in the twenty-four hours. The figure given by Beaumont, 1 
viz., 180 grammes pro die, based upon observations made upon the 
often-quoted Canadian hunter, Alexis St. Martin, is undoubtedly too 

1 Beaumont, Experiments and Observations on the Gastric Juice, Boston, 1834. 



216 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

low. The amount given by Bidder and Schmidt, 1 viz., that corre- 
sponding to about one-tenth of the body-weight, is probably more 
nearly correct. 2 It may be stated a priori, however, that the quantity 
secreted varies within wide limits, being influenced by numerous 
factors, and notably by the degree of the appetite and the amount and 
character of the food taken, especially that of the proteids. The 
age and sex of the individual, the time of day (notably in its relation 
to the ingestion of food), the emotions, etc., all influence the glandular 
activity of the stomach. 

From the non-digesting organ, as has been pointed out, from 1 to 
60 c.c. of gastric juice may be obtained at one time. The amount 
which can be procured during the process of digestion, on the other 
hand, varies with the amount of liquid ingested, the time of expres- 
sion, the size and motor power of the stomach, and the degree of 
transudation ; the process of resorption probably does not play any 
part, as it has been ascertained that very little water, if any, is 
absorbed in the stomach. 

According to Boas, from 20 to 50 c.c. of filtrate can normally be ob- 
tained exactly one hour after the ingestion of Ewald's test-breakfast. 3 

Abnormally large quantities of gastric juice are practically found 
only in cases of so-called hypersecretion, the " Magensaftfluss " of 
the Germans, which may occur periodically or continuously. For- 
merly the presence of appreciable quantities of gastric juice in the 
non-digesting organ was regarded as conclusive evidence of the 
existence of this condition, but in the light of Schreiber's researches 
this position can no longer be maintained. The diagnosis should, 
hence, only be made when in conjunction with the clinical symptoms 
of hypersecretion from 100 to 1000 c.c. of pure gastric juice can be 
obtained from the non-digesting organ. To this end, the stomach 
should be emptied completely by the tube, before retiring, and an 
examination made on the following morning, no food or liquids 
being allowed in the meantime. 

In various pathological conditions abnormally large quantities of 
liquid may be obtained, which cannot be regarded as gastric juice, how- 
ever. Attention will be drawn to these conditions at another place. 

CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 

Chemical Composition of the Gastric Juice. 

As has been briefly shown above, gastric juice consists of water, free 
hydrochloric acid, certain ferments and their zymogens, and mineral 
salts. Analyses giving the exact chemical composition of pure, un- 
con tarn mated gastric juice in man are wanting, owing to the difficulty 

1 Bidder u. Schmidt, Verdauungssafte u. d. Stoffwechsel, 1852. 

2 Griinewald's figure— i. e., 1580 grammes— I likewise regard as too low. According 
to my experience, the daily secretion appears to vary between 2000 and 3000 c.c. 

3 Eiegel, Die Erkrankungen des Magens, Part I. p. 88. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 217 

of excluding the saliva. In patients the subjects of gastric fistula 
analytical studies have, however, been made, and from the table 
below, taken from Schmidt, an idea may be formed of the various 
amounts of solid constituents contained in 1000 parts of gastric 
juice, uncontaminated by food or the products of digestion, but not 
free from saliva : 

Water 994.40 

Solids 5.60 

Organic material 3.19 

Sodium chloride 1.46 

Calcium chloride 0.06 

Potassium chloride 0.55 

Ammonium chloride . . 

Hydrochloric acid . . . 0.20 

Calcium phosphate ~\ 

Magnesium phosphate V 0.12 

Iron phosphate j 

The Acidity of the Gastric Juice is Referable to the 
Presence of Free Hydrochloric Acid. 

It has been conclusively demonstrated by Schmidt that the acidity 
of the gastric juice is due to the presence of free hydrochloric acid 



P.M. Fig. 44. 




















O 








•j-""^ "*** 


15 + '" ^^ 


i.o Si( ^ 


^EE 


S 


/ 


1.25 -7 




Z 


7 


y 


10 J- 


2 ± 




t ± 


J 






/ .■""" ""*, 


= y-=; 7 " : =-^___=___. = = = 






1/ s 




ji 




U.40 ? 


jr 


1 


r 





10 20 30 40 50 60 20 80 90 100 

Illustrating the curve of acidity after Ewald's test-breakfast. (Rosenheim.) 

-Lactic acid. X Beginning of the stage of free hydrochloric acid. 
The numbers upon the abscissa indicate the minutes. 



-Hydrochloric acid. - 
P. M. Pro mille 



After accurately determining the amount of chlorine and all basic 
substances present, it was found that after the latter had been satu- 



218 



THE GASTRIC JUICE AND GASTRIC CONTENTS. 



rated a quantity of hydrochloric acid still remained, which in the 
dog varied between 0.25 and 0.42 per cent., with an average of 0.33 
per cent. The amount of free acid was also determined by titration 
and the same results reached as by gravimetric analysis. 

While the acidity of pure gastric juice — i. e., gastric juice not 
contaminated with saliva or food in various stages of digestion — 
is thus solely due to the presence of free hydrochloric acid, other 
factors enter into consideration in the examination of the gastric 
contents during the process of digestion. Acid salts and varying 
amounts of lactic acid derived from the carbohydrates ingested are 

Fig. 45. 







































<•""" "n. 


25 **'" 5^ 


^" K * 


J^ V 


^ V 


S s^ 






S Ss - 




^ 






? 


/V- — - — — - 


10 -/ 2 ^- 




-t i 2 "**«- 


t -*• -•*. 




nn t-,<- 


0.5 ^2 


t r 


J-J 


1^ 


t _ _ __ 



30 



60 



90 



120 



T50 180 210 240 270 300 



Illustrating the curve of acidity after Riegel's test-meal. (Rosenheim.) 
-Hydrochloric acid. Lactic acid. X Beginning of the stage of free hydrochloric acid. 



then also found. At the beginning of digestion the acidity, accord- 
ing to Ewald, is due to a certain extent to the presence of lactic 
acid. 1 Hydrochloric acid, it is true, is present at the same time, 
but is held in combination by albuminous material. Later on, 
when the albuminous affinities have become saturated, it appears as 
such, with the result that the formation of lactic acid progressively 
diminishes, owing to the inhibitory action on the part of the hydro- 
chloric acid upon the lactic-acid-producing organisms. 2 

1 Ewald, Klin. d. Verdauungskrankheiten, 1890, vol. i. Ewald u. Boas, Beitr. z. 
Physiol, u. Path. d. Verdauung. Virchow's Archiv, 1885, vol. ci. p. 355, and 1886, 
vol. civ. p. 271. See Lactic Acid, p. 183. 

2 H. Strauss u. F. Bialocour, " Ueber d. Ahhangigkeit d. Milchsauregahrung v. 
HC1— Gehalt d. Magensaftes," Zeit. f. klin. Med., vol. xxviii. p. 567. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 219 

The varying degrees of acidity at different periods of digestion, 
after such test-meals as those of Ewald and Riegel, and the amount of 
the two acids present, may be seen from the accompanying diagrams 
(Figs. 44 and 45). 

Under pathological conditions the amount of free hydrochloric 
acid, as will be shown, may undergo great variations, diminishing on 
the one hand to zero, and increasing on the other to 0.5 per cent., or 
even more. At the same time the amount of lactic acid, which 
normally is present in very small amounts, and is absent altogether 
at the height of digestion, may greatly increase. Fatty acids, more- 
over, which are normally not present in the gastric juice, may then 
also be observed. It is thus seen that the total acidity of the gas- 
tric juice, especially in disease, cannot be regarded as indicating the 
amount of one single acid, unless the absence of other acids and 
acid salts is insured. 



Method of determining' the Total Acidity of the Gastric 

Contents. 

To this end, a known quantity of gastric juice is titrated with a 
one-tenth normal solution of sodium hydrate, using phenolphthalein 
as an indicator, when the number of cubic centimeters of the one- 
tenth normal solution employed, multiplied by the equivalent of 1 c.c. 
of this solution in terms of hydrochloric acid, will indicate the 
amount of acid present, from which the percentage-acidity is readily 
calculated. 

A normal solution of sodium hydrate is one containing the equiva- 
lent of its molecular weight in grammes — i. e., 40 grammes — in 
1000 c.c. of distilled water; a decinormal solution will, therefore, 
contain 4 grammes in the same volume of water. This quantity is 
dissolved in less than 1000 c.c. and the solution brought to the 
proper strength by titrating it with a solution of oxalic acid of 
known strength. 

From the equation 

2NaOfi + C 2 H 2 4 = C 2 Na 2 4 + 2H 2 0, 

it is seen that two molecules of NaOH (molecular weight 40) com- 
bine with one molecule of C 2 H 2 4 + 2H 2 (molecular weight 126), 
or 4 parts by weight of the former with 6.3 of the latter. One-tenth 
gramme of oxalic acid would hence require 15.873 c.c. of the one- 
tenth normal solution of NaOH for its neutralization, as is apparent 
from the equation 

6.3 : 1000 : : 0.1 : x ; 6.3a: = 100, and x = -gg = 15.673. 



220 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

One-tenth gramme of pure crystallized oxalic acid is dissolved in 
distilled water, and the solution titrated with the one-tenth normal 
solution of sodium hydrate, which is to be corrected, using two or 
three drops of a 1 per cent, alcoholic solution of phenolphthalein as 
an indicator, until the rose color of the solution has entirely disap- 
peared ; 15.9 c.c. should bring about this result. As the NaOH 
solution, however, has been purposely made too strong, less will be 
required. The amount of water that must then be added in order to 
bring the solution to its proper strength is determined by the formula 

Nd 
(J -= — , in which C represents the number of cubic centimeters of 
n 

water which must be added to the remaining solution, N the total 
number of cubic centimeters remaining after one titration, n the 
number of cubic centimeters consumed in one titration, and d the 
difference between the number of cubic centimeters theoretically 
required and that actually used in one titration. The solution hav- 
ing thus been properly diluted, the correctness of its strength is 
again tested and a further correction made, if necessary, until abso- 
lute accuracy has been attained. 

1000 c.c. of the one-tenth normal solution containing 4 grammes 
of NaOH are equivalent to 3.65 grammes of HC1, as is seen from 
the equation 

NaOH + HC1 = NaCl + H 2 

40 36.5 

1000 c.c. of the j 1 ^ normal solution represent 3.65 grammes of HC1 

100 " " " " " " 0.365 gramme " " 

10 " " " " " " 0.0365 " " " 

1 " " « " " represents 0.00365 " " " 

Application to the Gastric Juice. — Five or 10 c.c. of the filtered gas- 
tric juice are titrated with the one-tenth normal solution of sodium 
hydrate, using two or three drops of a 1 per cent, alcoholic solution 
of phenolphthalein, as an indicator, until the rose color which appears 
after the addition of every drop of the sodium hydrate solution no 
longer disappears on stirring or becomes deeper after the addition of 
a further drop. The number of cubic centimeters of the one-tenth 
normal solution employed multiplied by 0.00365 will then indicate 
the acidity of the 5 or 10 c.c. of gastric juice in terms of HC1, from 
which the percentage-acidity is calculated. 

Example. — Ten c.c. of gastric juice required the addition of 6.5 
c.c. of the one-tenth normal solution ; 6.5 X 0.00365 (i. e., 0.0237) 
would hence indicate the acidity of the 10 c.c. of gastric juice in 
terms of HC1, and 0.0237 X 10 = 0.237, the percentage-acidity. 

As these figures express the amount of HC1 in pure gastric juice 
obtained only from normal individuals, it has been found more con- 
venient for clinical purposes merely to indicate the degree of acidity 
bv the number of cubic centimeters of the one-tenth normal solution 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 221 

employed. In the above example, in which 6.5 c.c. were used, the 
percentage acidity woidd thus be indicated by the figure 65 — i. e. } 
the number of cubic centimeters of the one-tenth solution necessary 
to neutralize 100 c.c. of gastric juice. 

Under normal conditions figures varying from 40 to 60 are usually 
obtaiued one hour after the ingestion of Ewald's test-breakfast, 
while in pathological conditions greater variations are observed. 
In acute and chronic inflammatory conditions of the stomach, 
as well as in some of the neuroses, the acidity of the gastric contents 
is below normal. Higher figures are met with in cases of ulcer, in 
some cases of dilatation, and are especially frequent in some of the 
neuroses, in which a degree of acidity corresponding to 90 or even 
more is not infrequently observed. Increased acidity, usually asso- 
ciated with hypersecretion of gastric juice, is met with in the so- 
called hyperseeretio acida et coatinua of Eeichmann. 1 

It has been pointed out that the reaction of normal gastric juice 
is always acid, owing to the presence of free hydrochloric acid, and 
the same may be said to hold good for the gastric contents in general, 
obtained from a normal individual. Pathologically an acid reaction 
is also the rule, as in those cases in which hydrochloric acid is absent 
fatty acids and lactic acid usually make their appearance. It is, 
therefore, not surprising that an alkaline, neutral, or amphoteric 
reaction is but rarely, or at least not commonly, observed in the 
gastric contents artificially obtained, and practically seen only in 
the so-called mucous form of chronic gastritis, or in those rare cases 
of anadeny, iu which a complete destruction of the gastric glands 
has taken place. In vomited material, on the other hand, such 
observations are common, owing to the presence of large amounts 
of saliva. The vomited material in cases of so-called vomitus 
matutiiius, which is usually referable to a chronic catarrhal condition 
of the pharynx, generally presents an alkaline reaction, owing to 
the fact that the fluid brought up is largely unchanged saliva. 

Source of the Hydrochloric Acid. 

That the hydrochloric acid is not directly derived from the chlo- 
rides ingested is shown by the fact that it is secreted by starving 
animals. The same point is also proved by the observations of 
Sehreiber, which go to show that the secretion of the acid is con- 
tinuous, not to mention the well-known fact that even after the 
ingestion of material free from chlorine an acid gastric juice is 
secreted. It is apparent, then, that the chlorides of the blood must 
furnish the necessary chlorine, and as the pyloric glands, which con- 

1 Eeichmann. Berlin, klin. Woch., 1882, vol. xix. p. 606; 1854, vol. xxi. p. 768; 
1887, toI. xxiv. p. 12. 



222 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

tain no parietal cells, furnish an alkaline, and the fundus glands, 
which do contain parietal cells, an acid secretion, it is thought that 
these parietal cells are in some manner concerned in the production 
of the hydrochloric acid. The exact manner in which this takes 
place has not been definitely ascertained, but it is not improbable 
that the acid results from a " Masseneinwirkung " on the part 
of the carbonic acid, which is present in large quantities in the 
blood as such, upon the sodium chloride, and that owing to a specific 
action on the part of the parietal cells the hydrochloric acid is 
secreted into the ducts of the glands of the stomach, while the 
sodium carbonate which is formed at the same time returns to the 
blood. 

Two factors are thus necessary in order that a normal amount of 
hydrochloric acid should be secreted—?. <?., a normal condition of 
the blood and a normal condition of the cells. Whenever the 
integrity of either of these factors becomes impaired, it is clear 
that an abnormal secretion of hydrochloric acid or none at all will 
result. The nervous system, furthermore, must be taken into con- 
sideration as a third factor, as normal innervation is the sine qua 
non for the normal activity of any organ. The secretion of the 
acid is impaired whenever the nutrition of the cells of the stomach 
suffers, whether this be the result of inflammatory lesions, new 
growths, or hypersemic conditions of the stomach, the effect of 
renal, hepatic, or pulmonary diseases, etc., or in consequence of 
central or peripheral nerve influences. 

In the secondary dyspepsias, then, the result of renal, hepatic, 
cardiac, or hsemic diseases, etc., an examination of the gastric juice 
for free hydrochloric acid is of comparatively little value from a 
diagnostic standpoint, although it may suggest valuable points for 
the dietetic treatment of such patients. 

Significance of Free Hydrochloric Acid. 

Formerly it was believed that the principal function of the stomach 
was a digestive one, and that in the stomach, owing to the action of 
hydrochloric acid and pepsin, albumins were to a large extent 
transformed into peptones and albumoses. As pepsin is active only 
in the presence of a free acid, it was thought, moreover, that the 
power of the hydrochloric acid to render pepsin physiologically 
active constituted its entire field of usefulness. 

It had been noted over one hundred years ago, however, by the 
Abbe Spallanzani, that pieces of meat immersed in gastric juice 
resist the process of putrefaction for days. When it was shown, 
later on, that the free mineral acids are powerful antiseptics, and 
that the stomach secretes an amount of free hydrochloric acid 
sufficient to prevent the development of most of the putrefactive 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 223 

organisms, the time had come to doubt the correctness of the view 
previously held. 

Numerous experiments have been made in order to test the anti- 
septic and germicidal power of the gastric juice. Among the more 
important results achieved the following may be mentioned : the 
comma bacillus of cholera Asiatica is destroyed by normal acid 
gastric juice, while infection results when this has previously been 
neutralized. The same holds good for numerous other pathogenic 
organisms which are of special interest to the clinician. Among 
these may bo mentioned the various species of streptococcus, Staphylo- 
coccus pyogenes aureus, the bacillus of anthrax, etc. Unfortunately, 
however, not all species of pathogenic organisms are destroyed by the 
acid of the gastric juice, and the spores, moreover, of some of those 
that are destroyed are possessed of a considerable degree of resist- 
ance. This is especially true of the tubercle bacillus and in many 
cases of the spores of the anthrax bacillus. 

Those bacteria also which cause lactic acid and butyric acid fer- 
mentation resist the antifermentative power of the gastric juice to a 
certain extent, as may be concluded from the fact that they are 
always present in the intestines. At the beginning of the process of 
gastric digestion, when the hydrochloric acicl secreted is immediately 
taken up by the albuminous bodies present, traces of lactic acid can 
usually be demonstrated in the gastric contents if carbohydrates 
have been ingested. Later on, when free hydrochloric acid appears, 
lactic acid fermentation ceases. This observation is in accord with 
the fact that the action of the lactic acid producers is prevented by 
the presence of 0.7 pro mille of free hydrochloric acid. 

From what has been said it may be argued that as the principal 
function of the stomach consists in the furnishing of an antiseptic 
and germicidal fluid, under suitable conditions life could go on in 
the absence of the stomach. That this is possible has been demon- 
strated by Czerny, who succeeded in removing almost the entire 
organ from a dog. Five or six years later the same animal was 
killed in Ludwig's laboratory, and it was found at the autopsy that 
"near the cardia a small portion of the stomach had remained, 
surrounding a globular cavity filled with food." This dog then had 
lived for almost six years practically Avithout a stomach, had gained 
in Aveight, and was to all intents and purposes as healthy an animal 
as one provided Avith an entire organ. In the human being similar 
observations have been made on subjects of carcinoma of the stomach. 
It is thus very probable that the stomach, so far as the process of 
digestion is concerned, is not necessary for the maintenance of life. 

Literature. — Spallanzani, Experiences sur la digestion de 1'homme et de differ- 
entes especes d'animaux, Geneve, 1784. Bunge, Lehrbuch d. phvsiol. Chem., 1889, 
p. 44. Mester, " Ueber Magensaft u. Darmf aulniss," Zeit. f. klin. Med., vol. xxiv. p. 
441. Schmitz. " Zur Kenntniss d. Darmf aulniss." Zeit. f. physiol. Chem., vol. xvii. 
p. 401 ; " Die Beziehung d. Salzsaure d. Magensaftes z. Darmf aulniss," Ibid., vol. xix. 



224 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

p. 401. C. E. Simon, " On Indicanuria," Am. Jour. Med. Sci., 1895, vol. ex. p. 48. 
Czerny, Beitrage z. operativen Chirurgie, Stuttgart, 1878, p. 141. Ludwig u. Ogata, 
"Ueber d. Verdauung nacb d. Ausschaltung d. Magens," Du Bois' Arcbiv, 1883, p. 89. 
J. Carvallo u. Y. Pacbon, " Untersucliungen iiber d. Verdauung bei einem Hunde 
obne Magen," Arcb. der Pbysiol., 1894, p. 106. 

The Amount of Free Hydrochloric Acid. 

Pure gastric juice, according to Ewald, 1 Szabo, 2 and Boas, 3 con- 
tains from 2 to 3 pro mille of free hydrochloric acid. 

In the digesting organ such amounts are met with only at the 
height of digestion, and after all albuminous and basic affinities 
have been saturated. The time at which free hydrochloric acid can 
be demonstrated in the gastric contents after the ingestion of a meal 
will, hence, vary with the character of the food and its amount. 
When but little work is to be accomplished free hydrochloric acid 
is found much sooner than otherwise. After Ewald' s test-breakfast, 
for example, it appears after thirty-five minutes ; the point of maxi- 
mum acidity is reached after from fifty to sixty minutes, and corre- 
sponds to the presence of 1.7 pro mille. Following Sieger's meal, on 
the other hand, the free acid appears after one hundred and thirty- 
five minutes, and reaches its highest point (corresponding to 2.7 pro 
mille) in from one hundred and eighty to two hundred and ten 
minutes (Figs. 44 and 45). 

Clinically it is necessary to distinguish between euchlorhydria, or 
the secretion of a normal amount of free hydrochloric acid (0.1 to 
0.2 per cent.), hypochlorhydria, or the secretion of a deficient 
amount (less than 0.1 per cent.), hyperchlorhydria, in which more 
than 0.2 per cent, is found, and, finally, anachlorhydria, in which 
no hydrochloric acid at all is secreted. 

Euchlorhydria. — Euchlorhydria, when associated with clinical 
symptoms pointing to gastric derangement, is most commonly ob- 
served in nervous dyspepsia. A chronic gastritis can always be 
excluded in the presence of a normal amount of the free acid, thus 
constituting a most important point in the differential diagnosis 
between the two conditions. A normal secretion of free hydro- 
chloric acid is, furthermore, observed in some cases of atony or 
hypatony of the muscular walls of the stomach. 

Hypochlorhydria. — Hypochlorhydria is associated with all those 
diseases in which the secretory elements have been more or less 
damaged, as in subacute and chronic gastritis, in some cases of ulcer 
of the stomach or the duodenum, in incipient carcinoma, dilatation, 
and atony. 

Anachlorhydria. — Not many years ago it was thought that the 
absence of free hydrochloric acid from the gastric contents was 
pathognomonic of carcinoma of the stomach. This view was soon 
abandoned, however, as it was shown that cases of carcinoma occur 

1 Loc. cit. 2 D. Szabo, Zeit. f. pbysiol. Chem., 1877, vol. i. p. 155. 

3 Loc. cit. See also A. Scbiile, Zeit. f. klin. Med., 1896, vols, xxviii. and xxix. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 225 

in which hydrochloric acid is not only present, but present in ex- 
cessive amounts. This is true especially of those cases in which 
the malignant growth has started upon the base of an old ulcer. 
It was, furthermore, shown that anachlorhydria exists in almost all 
cases of advanced chronic gastritis, and is a very common occur- 
rence in neurasthenic and hysterical individuals, constituting the 
so-called hysterical anacidity. 

Hyperchlorhydria. — The existence of hyperchlorhydria is gen- 
erally indicative of a gastric neurosis, and is thus frequently met 
with in its simplest form in certain neurasthenic individuals. Asso- 
ciated with a continuous hypersecretion of gastric juice it constitutes 
the neurosis that has been described under the term hypersecretio 
acida et eoniinua. Hyperchlorhydria is also of frequent occurrence 
in cases of gastric ulcer, and may even occur in carcinoma, notably 
in those cases in which, as stated above, the new growth has started 
from an old ulcer. 

Test for Free Acids. 

Following a physical examination of the gastric contents, and, if 
acid, a determination of the total acidity, the next step will be to 
determine whether or not the acid reaction is referable to the pres- 
ence of a free acid, of combined acids, or of acid salts. 

The Congo-red Test. 1 — Congo-red is a carmin-colored powder, 
while its solutions are of a peach- or brownish-red color, which 
changes to azure blue upon the addition of a free acid, but remains 
unaffected in the presence of an acid salt. Congo-red may be em- 
ployed in solution or in the form of a test-paper. The latter, how- 
ever, is less delicate than the solution, and indicates only the pres- 
ence of 0:01 per cent, of hydrochloric acid, while a positive reaction 
can still be obtained with the aqueous solution in the presence of 
0.0009 per cent. The solution should be moderately dilute. The 
test-paper is prepared by soaking filter-paper, free from ash, in this 
solution, drying, and cutting it into suitable strips. In order to test 
for the presence of a free acid, it is only necessary to immerse a 
strip of the test-paper in the filtered gastric juice, or to add a drop 
or two of the solution to a small amount of the juice, when in the 
presence of a free acid a blue color will develop, which varies from 
a sky-blue to a deep azure according to the amount present. A 
negative result will exclude at once the possibility of peptic activity, 
as pepsin acts only in solutions containing a free acid. If the 
result of the test is positive, the nature of the free acid must 
still be ascertained, and it is, therefore, necessary to test for free 
hydrochloric acid, or in its absence for lactic acid and certain fatty 
acids. 

1 Eiegel, Deutsch. med. Woch., 1886, No. 35 ; and Boas, Diagnostik u. Therapie d. 
Magenkran kheiten. 

15 



226 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

Tests for Free Hydrochloric Acid. 

The various reagents which may be employed are given below, 
and are arranged according to their degree of delicacy, viz.: 

1. Dimethyl-amido-azo-benzol 0.02 pro mille 

2. Phloroglucin- vanillin 0.05 " 

3. Eesorcin 0.05 " 

4. Tropseolin 00 ' . . 0.30 " 

5. Mohr's reagent 1.00 " 

The Dimethyl-amido-azo-benzol Test. 1 — This test is known also 
as Topfer's test, and is destined to replace the older phloroglucin- 
vanillin and resorcin tests in the clinical laboratory. The delicacy 
of the reagent is such that the natural yellow color of the indicator 
is changed to a reddish tinge upon the addition of but one drop of 
a one-tenth normal solution of hydrochloric acid in 5 c.c. of dis- 
tilled water. Its superior delicacy, as compared with the phloro- 
glucin-vanillin and resorcin tests, is apparent from the fact that 5 
c.c. of a 0.5 per cent, solution of egg-albumin, to which six drops 
of a one-tenth normal solution of hydrochloric acid have been added, 
still give a positive reaction with dimethyl-amido-azo-benzol, while 
the phloroglucin-vanillin and resorcin reactions are negative. 
Organic acids, including lactic acid, yield a red color only when 
present in amounts exceeding 0.5 per cent. ; I have further ascer- 
tained that if albumoses are present, a cherry-red color is not obtained 
even though lactic acid be present to the extent of 1 per cent. Loosely 
combined hydrochloric acid and salts do not produce a red color. 

For practical purposes a 0.5 per cent, alcoholic solution is em- 
ployed. One or two drops of this are added to a small quantity 
of the gastric contents, which need not be filtered : in the presence 
of free hydrochloric acid a beautiful cherry-red develops at once 
which varies in intensity according to the amount of free acid 
present. In the presence of organic acids an orange color is 
obtained. In watery solution the color is a greenish yellow and the 
fluid is distinctly fluorescent. 

To extract the stomach contents with ether, before applying the 
dimethyl test, as has been suggested, will scarcely ever be necessary. 

I have personally used Topfer's test during the past nine years, 
and am well satisfied with the results. In teaching students it is 
well to show the color which one obtains with lactic acid in the 
presence of albumoses ; confusion as to whether or not free hydro- 
chloric acid is present will then not occur. 

The Phloroglucin-vanillin Test. 2 — The solution employed con- 
tains 2 grammes of phloroglucin and 1 gramme of vanillin, dissolved 
in 30 c.c. of absolute alcohol : a yellow color results, which gradu- 

1 Topfer, Zeit. f. physiol. Chem., 1894, vol. xix. Hari, Arch. f. Verdauungskrank. 
vol. ii. pp. 182 and 332. 

2 Gunzburg, Centralbl. f. klin. Med., 1887, vol. viii. No. 40. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 227 

ally turns a dark golden red, changing to brown when exposed to 
light. The solution should therefore be kept in a dark-colored 
bottle. Lenhartz suggests the use of separate solutions of phloro- 
glucin and vanillin, one or two drops of each being employed iii the 
test. Boas recommends a solution of the phloroglucin and vanillin, 
in the proportions indicated, in 100 grammes of 80 per cent, alcohol, 
and claims that the reagent is then still more sensitive and more 
stable. If a few drops of gastric juice, or even of the unfiltered 
gastric contents, containing 0.05 per cent, or more of free hydro- 
chloric acid, are treated with the same number of drops of the 
reagent, no change in color results, but upon the application of gentle 
heat — boiling and rapid evaporation are to be avoided- — a rose-tint or 
exceedingly fine rose-colored lines develop, which are characteristic 
of the presence of the free acid. 

For practical purposes it is best to carry on this slow evaporation 
on a thin porcelain butter-dish, the porcelain cover of a crucible, or 
in a small evaporating-dish of the same material. The color obtained 
in the presence of free hydrochloric acid is a rose color in every in- 
stance, and varies in intensity with the amount of acid present. A 
brown, brownish-yellow, or brownish-red color always indicates that 
excessive heat has been applied or that free hydrochloric acid is 
absent. 

Organic acids do not produce the reaction, nor is it interfered 
with by their presence, or that of albumins, peptones, or acid salts. 

A phloroglucin-vanillin test-paper, prepared by soaking strips of 
filter-paper, free from ash, in the solution and drying them, may 
also be employed. If a strip of this is moistened with a drop of 
gastric juice and gently heated in a porcelain dish, the rose color 
will develop in the presence of free hydrochloric acid, and does not 
disappear upon the addition of ether. 

The Resorcin Test. 1 — The solution consists of 5 grammes of 
resublimed resorcin and 3 grammes of cane-sugar, dissolved in 100 
grammes of 94 per cent, alcohol. It is equally as delicate as the 
phloroglucin-vanillin solution and has the advantage of greater 
stability. 

Five or six drops of gastric juice are treated with three to five 
drops of the reagent and slowly evaporated to dryness over a small 
flame, when a beautiful rose- or vermilion-red mirror will be obtained, 
which gradually fades on cooling. If the reagent is employed in 
the form of a test-paper, a violet color at first develops, which 
upon the application of heat turns brick red and does not disappear 
on treatment with ether. 

The presence of acid salts, organic acids, albumins, or peptones 
does not interfere with the reaction. 

1 Boas, Centralbl. f. klin. Med., 1888, vol. ix. No. 45. 



228 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

The Tropseolin Test. 1 — Tropseolin 00, when employed according 
to the method suggested by Boas, is a very reliable reagent, indi- 
cating the presence of 0.2 to 0.3 per cent, of free hydrochloric acid. 
Three or four drops of a saturated alcoholic solution of tropseolin 00, 
which has a brownish-yellow color, are placed in a small porcelain 
dish or cover, and allowed to spread over the surface. A like amount 
of gastric juice is then added and likewise allowed to flow over the 
surface of the dish ; upon the application of gentle heat beautiful 
lilac or blue stripes appear, which are said to be absolutely character- 
istic of free hydrochloric acid. 

A tropseolin test-paper may also be prepared by soaking filter- 
paper, free from ash, in the alcoholic solution, and then drying and 
cutting it into strips. A few drops of gastric juice containing free 
hydrochloric acid produce a more or less pronounced brown color 
upon this paper, which turns lilac or blue upon the application of 
gentle heat. Organic acids, when present in large amounts, likewise 
produce a brown color, but this disappears on heating, and a lilac or 
blue color does not result. 

For ordinary purposes this test is sufficient, and recourse need only 
be had to the more delicate reagents when a negative or a doubtful 
result is obtained. 

Mohr's Test, as modified by Ewald. 2 — Two c.c. of a 10 per cent, 
solution of potassium sulphocyanide are treated with 0.5 c.c. of a 
neutral solution of ferric acetate, and diluted to 10 c.c. with distilled 
water, a ruby-red solution resulting. Of this, a few drops are placed 
in a porcelain dish, when a drop or two of the filtered gastric con- 
tents are allowed to come into contact with the reagent. In the 
presence of free hydrochloric acid a light- violet color develops at the 
point of contact between the two fluids, and turns a deep mahogany- 
brown upon mixing. 

The test is not interfered with by the presence of acid salts or 
peptones, but is not so sensitive as those already described. 

The Benzopurpurin Test. 3 — Benzopurpurin 6B has been highly 
recommended by v. Jaksch as a very sensitive test for hydrochloric 
acid. It is best used in the form of a test-paper, prepared by soak- 
ing strips of filter-paper, free from mineral ash, in a concentrated 
watery solution of the reagent and allowing them to dry. 

In the presence of more than 0.4 gramme of hydrochloric acid 
in 100 c.c. of gastric juice the color of the test-paper immedi- 
ately turns a deep blackish-blue. Should a brownish-black color 
develop, this is likely due to the presence of organic acids, or, a mixt- 
ure of these and hydrochloric acid. If the color is caused by or- 

1 Ewald, Klinik d. Verdauungskrank., Berlin, 1888, vol. ii. ; and Boas, Deutsch. med. 
Woch., 1877, vol. xiii. p. 852. 

2 Ewald u. Boas, Virchow's Archiv, vol. ci. p. 325 ; vol. civ. p. 271. 

3 v. Jaksch, Klinische Diagnostik, 1896, p. 177. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 229 

ganic acids only, it will disappear on washing the strip with a little 
neutral ether, the original color of the test-paper being thus restored ; 
but if due to a mixture of the two, the reaction is less marked, and 
does not disappear. According to Hellstrom, 1 0.39 milligramme of 
hydrochloric acid, dissolved in 6 c.c. of water, can be recognized by 
the addition of only 5 milligrammes of benzopurpurin. 

Acid salts, peptones, and serum -albumin do not seriously inter- 
fere with the reaction. 

v. Jaksch claims that the benzopurpurin test-paper is more sensi- 
tive than the Congo-red paper. 

The Combined Hydrochloric Acid. 

It has been stated (see page 217) that the total acidity of the 
gastric juice can only be referred to hydrochloric acid when organic 
acids and acid salts are absent. But at the same time the free acid is 
titrated together with the loosely combined acid. The presence of free 
hydrochloric acid in normal amounts implies, of course, the existence 
of peptic activity, and indicates that all albuminous affinities have 
been saturated. In the absence of free hydrochloric acid, however, 
it is important to know whether or not hydrochloric acid is secreted 
at all — i. e., whether peptic digestion is at a standstill or whether an 
amount is secreted that is sufficient to saturate only certain albu- 
minous affinities without appearing in the free state. In the treat- 
ment of the various forms of gastric disease, more especially those 
associated with an absence of free hydrochloric acid, accurate knowl- 
edge in this respect is important. If no hydrochloric acid at all is 
secreted, the stomach can only be regarded as a storehouse, as it 
were, and proteids must be ordered in such a form that they may be 
subjected to the process of pancreatic digestion with as little delay 
as possible, the nutrition of the body being aided, if necessary, by 
a suitable administration of predigested food. If, on the other 
hand, an amount of hydrochloric acid is secreted which is sufficient 
to saturate the albuminous affinities of an ordinary meal, or at least 
of moderate amounts of proteids, the dietetic directions need not be 
so stringent. While in the former case the absence of loosely com- 
bined hydrochloric acid usually indicates complete destruction of 
the glandular elements of the stomach — in other words, an irrepar- 
able condition — a fair prognosis may be given when the amount of 
acid secreted is sufficient for the saturation of the albuminous 
affinities of an ordinary meal. The following table 2 shows the 
amount of hydrochloric acid necessary to saturate the affinities of 
known quantities of various articles of food, the figures given having 
reference to 100 c.c. or 100 grammes : 

1 Cited by v. Jaksch. 

2 Taken, in part from personal observations, and in part from Ehrlich, Dissert., 
Erlangen. 1893. 



230 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

Milk 0.32-0.56 gramme of pure HCI. 

Beef (boiled) 1.95-2.0 grammes " 

Mutton (boiled) 1.9 

Veal (boiled) 2.2 . " " " 

Pork (boiled) 1.5-1.6 " " " 

Sweetbread (boiled) ........ 0.9-0.95 gramme " " 

Calves' brains (boiled) 0.56-0.65 " " " 

Ham (raw) 1.9 grammes " li 

Ham (boiled) 1.3-1.8 " " " 

Flounder 1.41 « " " 

Liver sausage 0.8-0.9 gramme ". " 

Cervelat sausage 1.1 grammes " " 

Mettwurst 1.0 gramme " " 

Bologna sausage 1.49 grammes " " 

Blood sausage 0.3 gramme " " 

Potato (mashed) 0.48 " " " 

Rice (milk) 1.22 grammes " " 

Corn 0.27 gramme " " 

Graham bread 0.3 " " " 

Pumpernickel 0.7 " " " 

Wheat bread 0.3-0.5 « " " 

Rye bread ... 0.3-0.5 " " « 

Swiss cheese 2.6-2.7 grammes " " 

Fromage de Brie 1.3 " " " 

Edam cheese 1.4 " " " 

Roquefort cheese 2.1 " " (t 

Beer (German) 0.07-0.15 gramme " " 

Quantitative Estimation of the Hydrochloric Acid of the 
Gastric Juice. 

Tbpfer's Method. 1 — The free and combined hydrochloric acid is 
most conveniently estimated according to Topfer's method, which is 
both simple and sufficiently accurate for clinical purposes. 

In this method the total acidity (a) of a given amount of gastric 
juice — i. <?., the acidity referable to the presence of free hydrochloric 
acid, combined hydrochloric acid, acid salts, and any organic acids 
that may be present — is first determined (lactic acid and the fatty 
acids, if present, need not be removed), using phenolphthalein as an 
indicator. This is followed by a determination of the acidity refer- 
able to free acids and acid salts in the same amount of gastric juice 
(b), using alizarin (alizarin monosulphonate of sodium) as an indi- 
cator. As this does not react with loosely combined hydrochloric 
acid, the difference between a and b will indicate the amount 
of the latter. The free hydrochloric acid (c) finally is estimated with 
dimethyl-amido-azo-benzol as an indicator, the difference between a 
and b-\-c giving the acidity referable to organic acids and acid salts. 

The solutions required are the following : 

1. A decinormal solution of sodium hydrate. 

2. A 1 per cent, alcoholic solution of phenolphthalein. 

3. A 1 per cent, aqueous solution of alizarin. 

4. A 0.5 per cent, alcoholic solution of dimethyl-amido-azo-benzol. 
Three separate portions of 5 or 10 c.c. of filtered gastric juice are 

measured into three small beakers or porcelain dishes. To the 

1 Loc. cit. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 231 

first portion 1 or 2 drops of phenolphthalein are added, when it 
is titrated with the one-tenth normal solution of sodium hydrate. 
It is necessary, however, to titrate to the point of a deep red, and 
not to the rose hue which first appears. It will be seen that upon 
the addition of the first few drops of the one-tenth normal solution 
the red color, which first appears, disappears on stirring. Upon 
further titration a point is reached when this no longer occurs, and 
the color of the entire solution suddenly turns to a rose. This, 
however, is not the end-reaction that is desired. If the titration 
is continued, it will be observed that a dark-red cloud forms in the 
light rose-colored solution, which disappears on stirring ; finally, a 
point is reached when an additional drop no longer intensifies the color 
of the solution. This point is the end-reaction which must be reached. 
To the second portion 3 or 4c drops of the alizarin solution are 
added, when it also is titrated with the one-tenth normal solution of 
sodium hydrate until a pure violet color is obtained. As practice is 
required in order to determine this point with accuracy, Topfer 
advises to make previously the following simple tests : 

1. To 5 c.c. of distilled water add 2 or 3 drops of alizarin solu- 
tion, when a yellow color will result. 

2. To 5 c.c. of a 1 per cent, solution of disodium phosphate add 
the same number of drops, when a red or slightly violet color will 
be obtained. 

3. Five c.c. of a 1 per cent, solution of sodium carbonate, treated 
with 2 or 3 drops of the alizarin solution, will strike a pure violet ; 
this is the color to which the titration must be carried. 

In the third portion of the gastric juice the free hydrochloric acid 
is titrated, after the addition of 3 or 4 drops of the dimethyl-amido- 
azo-benzol, until the last trace of red — in the presence of free hydro- 
chloric acid — has disappeared, aud the color has become distinctly 
greenish yellow. The results are then calculated as in the following 
example : 

Ten c.c. of gastric juice, using phenolphthalein as an indicator, 
required 10 c.c. of the one-tenth normal solution in order to bring 
about the end-reaction, while a like amount titrated in the same 
manner with alizarin required 7 c.c. in order to bring about the 
same result. The difference between 10 and 7 — i. e., 3 — would 
thus indicate the number of cubic centimeters necessary to neutralize 
the amount of hydrochloric acid in combination with albuminous 
material. As 1 c.c. of the one-tenth normal solution represents 
0.00365 gramme of hydrochloric acid, the amount of acid thus held 
will be equivalent to 0.00365 X 3 = 0.01095 gramme of hydrochloric 
acid — i. e., 0.1095 per cent. 

In the estimation of the free hydrochloric acid, 2.3 c.c. of the one- 
tenth normal solution were required, using dimethyl-amido-azo-ben- 



232 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

zol as an indicator ; this would correspond to 0.00365 X 3.2 — i. e., 
0.1168 per cent. The value of the total acidity in terms of hydro- 
chloric acid is 10 X 0.00365 = 0.0365 gramme for every 10 c.c. of 
gastric juice, or 0.365 per cent. By deducting the amount of the 
free and combined hydrochloric acid, viz., 0.1095 -f 0.1168 = 0.2263, 
from this, it is found that the acidity of the gastric juice referable to 
organic acids and acid salts amounts to 0.1387 per cent., so that the 
results can be tabulated as follows : 

Free hydrochloric acid 0.1168 per cent. 

Combined hydrochloric acid 0.1095 " 

Organic acids and acid salts 0.1387 " 

Total acidity 0.3650 per cent. 

If free acid is absent, the deficit can be ascertained by titrating 
with decinormal hydrochloric acid, using dimethyl as an indicator. 

Estimation of Free Hydrochloric Acid (according to Sahli). — 25-30 
drops of Gunzburg's reagent are added to 10 c.c. of gastric j uice. 
The mixture is titrated with a decinormal sodium hydrate solution 
as usual until a drop of the mixture, warmed on the stirring-rod 
after each addition of the alkali, shows a red color. A porcelain 
dish can, of course, also be used, as in the qualitative test. 

The Method of Martius and Liittke (modified). 1 — This method 
is equally exact, but requires a greater expenditure of time. It is 
based upon the fact that upon incineration of the gastric juice 
the free hydrochloric acid and that loosely combined with albu- 
minous material escape, while the chlorine in combination with 
inorganic bases remains in the mineral ash unless a very intense 
heat is applied for some time. By subtracting the amount of chlorine 
present in the latter form from the total amount, the quantity in 
combination with albuminous material and that occurring as free 
acid will be found. The total acidity of the gastric juice is then 
determined, and that referable to the presence of the free and com- 
bined hydrochloric acid subtracted, the difference giving the amount 
of organic acids present. By determining the acidity due to the pres- 
ence of free hydrochloric acid according to Topfer's method, and 
deducting the amount found from that referable to the presence of free 
and combined hydrochloric acid, the amount of the latter is obtained. 

Reagents required : 

1. A solution of silver nitrate in nitric acid of such strength that 
1 c.c. shall represent 0.00365 gramme of hydrochloric acid. 

2. Liquor ferri sulphurati oxydati. 

3. A decinormal solution of ammonium sulphocyanide. 

4. A one-tenth normal solution of sodium hydrate. 

5. A 1 per cent, alcoholic solution of phenolphthalein. 

1 F. Martius u. L. Liittke, Die Magensaure des Menschen, Stuttgart, 1892. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 233 

6. A 0.5 per cent, alcoholic solution of dimethyl-aniido-azo-benzol. 
Preparation of the solutions : 

1. The silver nitrate solution. As a solution is required of such 
strength that 1 c.c. shall be equivalent to 0.00365 gramme of hydro- 
chloric acid, the amount of silver nitrate that must be dissolved in 
1000 c.c. of water is ascertained in the following manner: since 
169.66 (molecular weight) parts by weight of silver nitrate combine 
with 36.5 parts of hydrochloric acid (molecular weight), the amount 
of silver nitrate required for each cubic centimeter is found from the 
equation 

169.66 : 36.5 : : x : 0.00365 ; 36.5 x = 0.6192590 ; x = 0.0169. 

In 1 c.c. of the silver solution 0.0169 gramme of silver nitrate must 
thus be present, or 16.9 grammes in the liter. This quantity, or 
roughly 17 grammes, is weighed off and dissolved in 900 c.c. of a 
25 per cent, solution of nitric acid ; as the acid must be present in 
excess, the solution is purposely made too strong. To this solution 
50 c.c. of the liquor ferri sulphurati oxydati are added. The solu- 
tion is then brought to the proper strength by titration of a known 
number of cubic centimeters of a one-tenth normal solution of 
hydrochloric acid and correcting as usual. 

2. The ammonium sulphocyanide solution. A normal solution of 
ammonium sulphocyanide contains 75.98 grammes (molecular 
weight) per liter, and a decinormal solution 7.598 grammes. This 
quantity, or roughly 8 grammes, is dissolved in about 900 c.c. of 
water and the solution brought to the proper strength by titrating a 
known number of cubic centimeters of the silver nitrate solution, 
Avhen each cubic centimeter should correspond to 1 c.c. of the silver 
solution — i. e., to 0.00365 gramme of hydrochloric acid. It is 
corrected as described elsewhere. 

Method. — 1. To determine the total amount of chlorine present : 
10 c.c. of filtered gastric juice — Martius and Liittke make use of 
the unfiltered gastric contents — are measured into a small flask 
bearing a 100 c.c. mark, and treated with an excess of the one-tenth 
normal solution of silver nitrate. Experience has shown that 20 c.c. 
are sufficient. The mixture is agitated and allowed to stand for ten 
minutes. Distilled water is then added to the 100 c.c. mark; the 
mixture is agitated once more and filtered through a dry filter into 
a dry beaker. Fifty c.c. of the filtrate are titrated with the one- 
tenth normal, solution of ammonium sulphocyanide until the blood- 
red color which appears upon the addition of every drop — due to 
the formation of ferric sulphocyanide — no longer disappears on 
stirring. By multiplying the number of cubic centimeters of the 
ammonium sulphocyanide solution used by 2 (the number of cubic 
centimeters that would have been necessarv for the precipitation of 
the excess of silver in 100 c.c.) and deducting the result from the 



234 THE GASTRIC JUICE AND 'GASTRIC CONTENTS. 

number of cubic centimeters of the one-tenth normal solution of 
silver nitrate employed, viz., 20, the number of cubic centimeters 
of the latter solution is found which was necessary to precipitate 
the chlorine in 10 c.c. of the gastric juice. As 1 c.c. of the solu- 
tion represents 0.0036 gramme of hydrochloric acid, it is only nec- 
essary to multiply this figure by the number of cubic centimeters 
used in precipitation of the chlorine. The resulting value. T y 
expresses the total amount of chlorine present. 

As a general rule, it is not necessary to decolorize the gastric 
juice. If desired, however, 5 to 15 drops of a 5 per cent, solution 
of potassium permanganate may be added to the 10 c.c. employed, 
after the mixture has stood for ten minutes. 

2. Determination of the amount of chlorine in combination with 
inorganic bases, F. Ten c.c. of the filtered gastric juice are 
carefully evaporated to dryness in a platinum crucible, on a water- 
bath or upon a plate of asbestos, in order to avoid sputtering (as the 
heat applied in the process of incineration is not very intense, a 
porcelain crucible may also be employed). The residue is then care- 
fully incinerated over an open flame, the process being carried only 
to the point when the organic ash no longer burns with a luminous 
flame. Intense heat should be avoided, as the chlorides are volati- 
lized upon the application of red heat. On cooling, the ash is 
moistened with a few drops of distilled water and mixed with a 
stirring-rod, when the residue is extracted in separate portions with 
100 c.c. of hot distilled water and filtered. This amount is usually 
sufficient to dissolve all the chlorides present. If any doubt should 
exist, however, it is only necessary to add a drop of the silver solu- 
tion to a few drops of the last portion of the filtrate : the formation 
of a cloud, referable to silver chloride, will necessitate still further 
washing. The whole filtrate is then treated with 10 c.c. of the one- 
tenth normal solution of silver nitrate, and the amount consumed 
in the precipitation of the chlorides determined by titration with 
the one-tenth normal solution of ammonium sulphocyanide, as de- 
scribed above. The hydrochloric acid present in combination with 
inorganic bases is thus determined. The difference between the 
amount of hydrochloric acid in combination with inorganic bases 
and the total amount of chlorine in terms of hydrochloric acid will 
then indicate the amounts of the free and of the combined hydro- 
chloric acid, which are termed L and Q respectively; hence 
T—F=L+ C. 

3. The total acidity in terms of hydrochloric acid is further de- 
termined according to the method given elsewhere (see page 157) and 
indicated by the letter A. The difference between the total acid- 
ity and the amount of free and combined hydrochloric acid will 
represent the amount of organic acids and acid salts, O ; hence 
= A-(L+C). 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 235 

The free hydrochloric acid finally is determined according to the 
method of Topfer. The difference between the value thus found 
and that expressing the amount of free and combined hydro- 
chloric acid will indicate the amount of the latter ; hence (L -f- C) 

— l = c. 

Leo's Method. 1 — This method is based upon the observation that 
calcium carbonate combines with free and combined hydrochloric acid 
at ordinary temperatures to form neutral calcium chloride, while the 
acid phosphates are not affected. It is thus clear that by determin- 
ing the total acidity of the gastric juice, and deducting from this 
the acidity referable to acid salts, the amount of the physiologically 
active hydrochloric acid — i. e., of the free and combined hydrochloric 
acid — is obtained. 

As it has been shown that in the presence of calcium chloride 
(formed, as indicated above, upon the addition of calcium carbonate), 
owing to the formation of calcium monophosphate — CaHP0 4 , twice 
the quantity of sodium hydrate is taken up, it is necessary to make 
the first titration also after the addition of an excess of calcium 
chloride. 

Reagents required : 

1. A one-tenth normal solution of sodium hydrate. 

2. A 1 per cent, alcoholic solution of phenolphthalein. 

3. A concentrated solution of calcium chloride. 

4. Chemically pure calcium carbonate. The purity of the salt 
may be tested by stirring a small piece with water ; the solution 
should not color red litmus-paper blue. A solution of the salt in 
dilute hydrochloric acid should not yield a precipitate when treated 
with sulphuric acid. 

Method. — Organic acids that may be present are first removed 
by shaking with ether, 50 to 100 c.c. being required for each 10 c.c. 
of gastric juice. The total acidity of the gastric juice is then de- 
termined in 10 c.c. of the filtered liquid after the addition of 5 c.c. 
of the concentrated solution of calcium chloride, the result being 
termed A. 

The acidity referable to the presence of acid phosphates is deter- 
mined as follows : 15 c.c. of filtered gastric juice are treated with a 
pinch of dry and chemically pure calcium carbonate ; the mixture is 
thoroughly stirred, and passed at once through a dry filter. Ten 
c.c. of the filtrate, from which the carbon dioxide is expelled by 
means of a current of air, are then treated with 5 c.c. of the 
calcium chloride solution and titrated as above, the resulting value 
being termed P. A — P is hence equivalent to L -f C. The 
value of C can then be ascertained by determining the acidity 
referable to free hydrochloric acid according to Topfer' s method, and 
deducting the value found from Z+ C. 

1 Leo, Centralbl. f. d. med. Wiss., 1889, vol. xxvii. p. 481. 



236 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

This method is sufficiently accurate for practical purposes, and has 
the advantage of not requiring the expenditure of much time. 

The Ferments of the Gastric Juice and their Zymogens. 

Pepsin and Pepsinogen. — According to our present knowledge, 
the zymogen of pepsin, viz., pepsinogen or propepsin, and not pepsin 
itself, is secreted by the chief cells of the fundus glands. This view 
is based upon the observation that an aqueous extract of the mucous 
membrane of the stomach of a fasting animal recently killed does 
not lose its digestive power for a considerable length of time when 
treated with a 1 per cent, solution of sodium carbonate at a tempe- 
rature of from 38° to 40° C, whereas pepsin itself is thus rapidly 
destroyed. It is natural then to conclude that the glands of the 
stomach do not contain pepsin, but some other substance during the 
process of fasting, which is capable of resisting the action of sodium 
carbonate, and which can be transformed into pepsin by the addition 
of hydrochloric acid. This substance has been termed pepsinogen or 
projiepsin. As a rule, pepsin is obtained only from the mucous 
membrane of the digesting organ, while at other times the physio- 
logically inactive zymogen is found. As the zymogen, moreover, is 
probably always present together with pepsin in the gastric juice 
obtained from healthy individuals during the process of digestion, it 
is not clear whether the transformation of the zymogen into its fer- 
ment takes place in the body of the cell or after secretion. There is 
evidence to show, however, that the latter view is correct. 1 

This is not the place to enter into a detailed consideration of the 
various properties of pepsin, and it will suffice to say that the activity 
of the ferment is destroyed by even very dilute solutions of the 
alkaline carbonates. The same result is reached by exposing a watery 
solution of pepsin to a temperature of 70° C, while in a dry state 
a temperature of 100° C. will not destroy its activity ; this is shown 
by the fact that a specimen of pepsin thus treated is, on cooling, still 
capable of digesting albumins in the presence of hydrochloric acid. 

While pepsin is capable of digesting albumins in the presence of 
other acids, viz., phosphoric, sulphuric, oxalic, acetic, lactic, and 
salicylic acids, the solutions must be stronger than in the case of 
hydrochloric acid. With lactic acid, for example, a satisfactory 
result is reached only with a concentration of from 12 to 18 pro mille, 
while of hydrochloric acid 2 to 4 pro mille are sufficient. Larger or 
smaller amounts do not act so promptly. 

Very important from a practical standpoint is the fact that but 
small quantities of pepsin are required to digest large amounts of 
albumin. Petit 2 thus claims that a pepsin preparation from his 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. 

2 Petit, " Etude sur les ferments digestifs," Jour. de. Therap., 1880. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 237 

laboratory was capable of dissolving 500,000 times its weight of 
fibrin in seven hours. This property possessed by pepsin, of doing 
an amount of work that is widely out of proportion to the amount 
of ferment present, is common to all ferments, and is dependent upon 
the fact that the ferment itself undergoes no change during the 
process. 

Figures expressing the exact quantity of pepsin or of its zymogen 
produced in the twenty -four hours are lacking, and inferences can 
hence only be drawn as to the physiological activity of the ferment 
from the rapidity with which given amounts of albuminous material 
are digested. This, however, depends to a large extent upon the 
nature and concentration of the free acid present. Under normal 
conditions 25 c.c. of gastric juice will dissolve 0.05 to 0.06 gramme 
of serum-albumin in one hour, the same amount of coagulated egg- 
albumin in three hours, and a like amount of fibrin in one hour and 
a half. 

As abnormalities in the circulation and innervation of the stomach 
apparently do not influence the production of pepsin, or rather of its 
zymogen, a diminution in the degree of peptic activity, or its total 
absence, may be referred directly to disease of the stomach itself, 
viz., its glandular apparatus. The determination of the presence or 
absence and relative amount of pepsin in the gastric juice, hence, 
furnishes more useful information than the recognition of the presence 
or absence of free hydrochloric acid. 

As pepsin is formed from pepsinogen through the agency of a free 
acid, its presence, in the absence of organic acids in notable quan- 
tities, indicates at once the presence of hydrochloric acid. It may 
be said, vice versa, that if free hydrochloric acid is present in the 
gastric juice, and the latter digests albumins, pepsin also will be 
found. Should the zymogen alone be present, digestion will take 
place only upon the addition of an acid, while an absence of diges- 
tion upon the addition of hydrochloric acid indicates the absence of 
both pepsin and its zymogen. At times, though rarely, a "gastric 
juice " is met with which is capable of digesting albumin in the 
absence of hydrochloric acid, owing to the presence of pancreatic 
juice — a point which is important, both from a diagnostic and a 
prognostic point of view. 

In the differential diagnosis of a chronic gastritis and a neurosis, 
or a dyspeptic condition referable to hyperemia of the gastric mucous 
membrane, the demonstration of the presence of the zymogen in the 
absence of hydrochloric acid may, at times, be very important, bear- 
ing in mind the fact that circulatory and nervous disturbances 
apparently do not influence the production of pepsinogen. An entire 
absence of the latter would, of course, warrant the diagnosis of 
complete anadeny of the stomach. 



238 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

Tests for Pepsin and Pepsinogen. — Test foe the Enzyme. — If 
the presence of free hydrochloric acid has previously been ascertained, 
25 c.c. of filtered gastric juice are set aside and kept at a tempera- 
ture of from 37° to 40° C, a bit of coagulated egg-albumin, fibrin, 
or serum-albumin being added. In order to permit of a comparison 
of results, the same amounts should always be taken ; 0.05 to 0.06 
gramme of egg-albumin, as has been shown, ought, under physiolog- 
ical conditions, to be digested in three hours. 

Test for the Zymogen. — Should hydrochloric acid be absent, 
the .test is made in the same manner, after the addition of from 3 to 
5 drops of the officinal solution of hydrochloric acid to 25 c.c. of the 
filtrate. Under such conditions usually pepsinogen alone is found. 

Quantitative Estimation. — Of Pepsin. — Accurate methods for 
the quantitative estimation of pepsin are unknown, and relative 
values only can be obtained. 

Hammer schlag' 's Method. 1 — Three Esbach's tubes (albuminimeters) 
are employed. Tube A is filled to the mark U with a mixture of 
10 c.c. of a 1 per cent, solution of serum-albumin in 0.4 per cent, 
of hydrochloric acid and 5 c.c. of filtered gastric juice. The second 
tube, B, which is the standard, is likewise filled to the mark U, but 
0.5 gramme of pepsin is added to the serum solution, instead of the 
gastric juice. The third tube, C, contains merely a mixture of the 
serum solution and 5 c.c. of water. After the tubes have been kept 
in the thermostate for one hour, at a temperature of 37° C. Esbach's 
reagent is added to each tube to the mark R. After standing for 
twenty-four hours the amount of precipitated albumin is read oif, 
and the difference between that in tube A and tube C compared with 
that in tube B. 

Mett's Method. 2 — Satisfactory comparative results can also be 
obtained with the method suggested by Mett. Capillary glass tubes 
are prepared measuring from 1 to 2 mm. in diameter. They are 
filled with white of egg, which is coagulated in the tubes at a tem- 
perature of 95° C. The tubes are then cut into pieces from 1 to 2 
cm. long and placed in the digestive mixture to be examined. The 
length of the column digested in a given length of time serves as a 
measure of the digestive power of the specimen examined. In 
practice this column should be measured in millimeters with the 
aid of a magnifying-glass. The calculation of the corresponding 
amount of ferment is based upon the law of Schiitz and Borissow, 
viz., that the corresponding amounts of ferment in two solutions 
bear the same ratio toward each other as the square of the number 
of millimeters of the column of egg albumin which has been dis- 
solved in the same length of time. 

1 Hammerschlag, Wien. med. Presse, 1894, vol. xxxv. p. 1654. 

2 Mett's method is described by Pawlow, die Arbeit d. Verdauungsdriisen. Trans- 
lated into German from the Russian by A. Walther, Wiesbaden, 1898. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 239 

Example. — The gastric juice of a normal individual is procured 
at the height of digestion after giving Ewald's test breakfast. The 
tube is digested for thirty minutes ; at the end of this time the 
height of the column of albumin digested measures 3 mm. Then 
the stomach contents of a second individual are obtained (the 
patient's) and similarly treated ; in this case the column of digested 
albumin measures 2 mm. The corresponding amounts of ferment 
are then as 9 is to 4. 

Of Pepsinogen. — In order to estimate the amount of pepsinogen 
the method -of Boas may be employed. To this end, the gastric 
juice is diluted with distilled water in varying proportions, such as 
1 : 5, 1 : 10, 1 : 20, etc. A known quantity of coagulated albumin 
is added to each specimen, as also 1 or 2 drops of an officinal 
solution of hydrochloric acid, for each 10 c.c. employed. These 
tubes are kept at a temperature of from 37° to 40° C, when the 
degree of dilution is noted at which the bit of egg-albumin is still 
dissolved. The greater the degree of dilution at which digestion 
still takes place, the greater the amount of pepsin or of its zymogen 
present. 

Both Hammerschlag's and Mett's method can also be applied in 
the estimation of pepsinogen, after rendering the gastric contents 
acid, with hydrochloric acid, to the extent of from 1 to 2 pro mille. 

If it is desired to exclude definitely the presence of pepsin and 
pepsinogen in the stomach, the method of Jaworski should be em- 
ployed. To this end, about 200 c.c. of a decinormal solution of 
hydrochloric acid are poured into the stomach through a tube and 
aspirated after one-half hour. If the fluid removed contains no 
pepsin, the absence of both the enzyme and its zymogen may be 
inferred. 

The Milk-curdling Ferment and its Zymogen, viz., Chymosin 
and Chymosinogen. — A great deal of what has been said above 
regarding pepsin and its zymogen also holds good for chymosin and 
its pro-enzyme. The pro-enzyme thus also appears to be formed by 
the cell, as a neutral aqueous extract of the mucous membrane of the 
stomach does not, as a rule, contain the ferment, but the zymogen, 
the ferment resulting only when the latter is treated with a free acid, 
It differs from pepsin in that it can exert its physiological activity 
in feebly acid, neutral, and even feebly alkaline solutions. Exposure 
of an active solution of chymosin to gastric juice containing 3 pro 
mille of free hydrochloric acid, moreover, at a temperature of from 
37° to 40° C, leads to its destruction. 

Its specific action is exerted upon milk, or lime-containing solu- 
tions of casein, which are coagulated in neutral or feebly alkaline 
solutions. 

In this connection it is important to note that the addition of a 
few cubic centimeters of a solution of calcium chloride, or any other 



240 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

soluble lime salt, results in a transformation of the zymogen into the 
physiologically active ferment, and that hydrochloric acid, while it 
normally causes such transformation, is not absolutely necessary in 
the presence of calcium chloride. 

Under physiological conditions chymosin and its zymogen are 
always present in the gastric juice. In disease the inferences that 
may be drawn from a quantitative estimation of the ferment and its 
zymogen have been well formulated by Boas, 1 to whom we are espe- 
cially indebted for a great deal of valuable information in this con- 
nection : 

1. Notwithstanding the absence of free hydrochloric acid, chymo- 
sin may be present, although in minimal traces — i. e., demonstrable 
with a dilution of from 1 : 10 to 1 : 20 (see method below). 

2. In the absence of free hydrochloric acid the zymogen may still 
be present in normal amounts — i. e., demonstrable with a dilution 
of from 1 : 100 to 1 : 150. The presence of the zymogen, especially 
when repeatedly observed, probably always permits of the conclusion 
that we are not dealing with an organic disease of the stomach, but 
with a neurosis or a hypersemic condition of the mucous membrane 
referable to disease of other organs. 

3. The zymogen may occur in moderately diminished amount, 50 
per cent, only being present. This is usually owing to the existence 
of a gastritis which has not reached its highest degree of severity. 
The nearer the amount of zymogen approaches the normal, the 
greater will be the probability of an ultimate recovery under suit- 
able treatment. 

4. The amount of the zymogen is greatly diminished (dilutions 
of 1 : 10 to 1 : 25 yielding a negative result) or may be absent alto- 
gether. In cases of this kind a severe and usually incurable gas- 
tritis exists, either primary or occurring secondarily to carcinoma, 
amyloid degeneration, etc. 

5. In conditions 1, 2, and 3, the re-establishment of the secretion 
of hydrochloric acid may be attempted with some prospect of success 
by means of stimulating remedies. 

These conclusions are based upon the employment of Ewald's 
test-breakfast, and cannot be applied to observations made after 
other test-meals, without previous studies in this direction. 

Testing for the presence of chymosin and its zymogen, moreover, 
is of decided value in cases in which alkaline material is vomited, 
and where we may be called upon to decide whether this contains 
constituents of the gastric juice or not. 

Tests for Chymosin and Chymosinogen. — Test for the En- 
zyme. — Five to 10 c.c. of milk are treated with from 3 to 5 
drops of the filtered gastric juice and kept at a temperature of from 

1 Boas. Centralbl. f. d. med. Wiss., 1887, vol. xxv. p. 417; and Zeit. f. klin. Med., 
1888 : vol. xiv. p. 240. See also J. Friedenwald, Med. News, 1895. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 241 

37° to 40° C. for ten to fifteen minutes. If coagulation occurs 
during this time, it may be concluded that the enzyme is present. 

Test foe the Zymogen. — The milk is treated with 10 c.c. of 
the filtered and feebly alkalinized gastric juice and with 2 or 3 c.c. 
of a 1 per cent, solution of calcium chloride. The mixture is kept 
at a temperature of from 37° to 40° C, when in the presence of 
the zymogen the formation of a thick cake of casein will be observed 
to occur within a few minutes. 

Quantitative Estimation. — Of the Enzyme. — The method is 
based upon the fact that on gradually diluting a specimen of gastric 
juice a point is finally reached at which a chymosin reaction can no 
longer be obtained, the value being, of course, a relative one. Under 
physiological conditions a positive reaction can still be observed with 
a degree of dilution varying between 1 : 30 and 1 : 40. 

The gastric juice is neutralized with a very dilute solution of 
sodium hydrate. Tubes are then prepared containing from 5 to 10 
c.c. of the gastric juice, diluted in the proportion of 1 : 10, 1 : 20, 
1 : 30, etc., to which an equal amount of neutral or amphoteric milk 
is added. The tubes, properly labelled, are kept at a temperature 
of from 37° to 40° C, and the degree of dilution noted at which 
coagulation still occurs. 

Of the Zymogen. — The gastric juice is rendered feebly alkaline 
and tubes are prepared containing equal amounts of milk and 
gastric juice, the latter variously diluted, as above directed ; the 
examination is then carried on in the same manner. Normally a 
positive reaction is obtained with a dilution varying between 1 : 150 
and 1 : 100. Allowance must, of course, be made for the amount 
of fluid which is added during the process of neutralization. 

The Products of Gastric Digestion. 

Digestion of the Native Albumins. — The first step in the proc- 
ess of albuminous digestion in the stomach is one of swelling, 
which may be observed when a flake of fibrin, for example, is placed 
in gastric juice and the temperature maintained between 37° and 
40° C. Very soon simple solution takes place, which is followed 
by the process of " denaturization," as Neumeister terms it, in 
which the native albumins are transformed into acid albumins or 
syntonins, owing to the continued activity of the hydrochloric acid 
and pepsin. The pepsin, however, acts only as an adjuvant to the 
acid, and hydrochloric acid alone is capable of effecting the same 
result. But while in the absence of pepsin more concentrated solu- 
tions of the acid and a higher temperature are required, the tem- 
perature of the body and the amount of hydrochloric acid secreted 
by the stomach are sufficient when pepsin is present. Pepsin, in 
the absence of free hydrochloric acid, is perfectly inert. 

The " denaturization " of the native albumins is followed by a 

16 



242 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

splitting up of the albuminous molecule and a process of hydration, 
the so-called primary albumoses being the first products thus formed. 
During the further process of digestion the deutero-albumoses then 
result, and from these the peptones, to which, in contradistinction 
to the peptones formed during the process of pancreatic digestion, 
the term amphopeptone has been applied by Kuhne. 

Digestion of the Proteids. — The digestion of casein, which 
belongs to the class of nucleo-albumins, differs from the process 
described. The casein of the milk is present in solution as a neutral 
calcium salt, and as it has the character of a polybasic acid, calcium 
chloride and the corresponding acid casein salt will result in the 
presence of the hydrochloric acid of the stomach ; still later, when 
more hydrochloric acid has been secreted, insoluble casein, as such, 
will be found. While the acid is thus capable of causing the pre- 
cipitation of casein, it has also been shown that the same result may 
be reached in the absence of hydrochloric acid. According to 
Hammarsten, this is brought about in consequence of the hydrolytic 
action on the part of the chymosin, the calcium salt of paracasein 
(cheese), and a small amount of an album ose-like posset-albumin 
being formed. This latter process is now supposed to take place in 
the stomach after the hydrochloric acid has previously transformed 
the neutral into the acid casein salt. When this stage is reached the 
paracasein is decomposed into an albumin and an insoluble nuclein. 
The albumin is then further digested as described ; a hetero-albumose, 
however, does not result. The remaining proteids, such as haemoglo- 
bin, glucosides, etc., are similarly acted upon by the gastric juice, and 
are first split up into the corresponding albumins and their pairlings. 
The albuminous radicles are then digested, as described. 

Digestion of the Albuminoids. — Of the albuminoids, only col- 
lagen and elastin undergo digestion in the stomach, gelatoses and 
elastoses being formed during the process, while keratin passes off 
undigested. Hetero-albumoses, however, are formed from neither 
collagen nor elastin, but merely proto-albumoses, which in turn are 
transformed into deutero-albumoses, and these into peptone. 

Digestion of the Carbohydrates.-^-The secretion of the stomach 
itself is not capable of digesting carbohydrates. There appears to 
be no doubt, however, that a transformation of starches into sugar 
takes place during the earlier stages of digestion. This is owing to 
the continued action of the ptyalin of the saliva (see page 199) in 
the stomach, which proceeds until the amount of hydrochloric acid 
secreted reaches 0.01 per cent, or more, it being remembered that the 
transformation of starches into sugar takes place best in a neutral 
or feebly alkaline medium. 

The question whether or not a diastatic ferment occurs in the 
mucus secreted by the stomach itself is unimportant, as cases have 
but rarely been observed in which there was an absence of ptyalin 
from the saliva. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 243 

As indicated in the chapter on the Saliva, a number of intermediary 
products are formed in the transformation of starch into sugar, of 
which an idea may be had from the accompanying table : 

Starch. 

Amidulin. 

I 

I I 

Erythrodextrin. Maltose. 

Achroodextrin a Maltose. 

I . I 

Achroodextrin /? Maltose. 

I ! 

Achroodextrin y (maltodextrin) Maltose. 

I I 

Maltose. Maltose. 

In the mouth this transformation is effected very rapidly in the 
case of certain starches, such as corn-starch and rye-starch, and it is 
possible to demonstrate the presence of sugar after from two to six 
minutes. Potato-starch, on the other hand, requires a much longer 
time, viz., from two to four hours. This difference is entirely de- 
pendent upon the varying degree of resistance offered to the action 
of the saliva by the enclosing envelope of cellulose, as is apparent 
from the fact that a paste made from potatoes is digested just as 
rapidly as one made from rye. 

For practical purposes, the digestion of carbohydrates in the 
stomach may be disregarded as insignificant. 

Fats are not digested in the stomach. 

From the above considerations it is apparent that under physio- 
logical conditions a mixture of various products is met with in the 
stomach at the height of digestion, and it might be expected that 
from a preponderance of the one over the other definite and valuable 
conclusions as to the digestive power of the organ could be reached. 
While this is true in a certain sense, the quantitative methods of 
analysis that would have to be employed in order to obtain definite 
data are as yet too complicated for the purposes of the clinician, and 
from the simple qualitative tests not much information can be de- 
rived. The recognition of the presence of peptones would thus 
merely indicate the presence of hydrochloric acid and pepsin in a 
general way, as peptones may be formed in the absence of hydro- 
chloric acid and in the presence of organic acids, which may be found 
in pathological conditions. A portion of the albumin of milk, eggs, 
meat, etc., is, moreover, already peptonized during the process of 
boiling. It is not surprising then that peptones may be demonstrated 
in practically every specimen of gastric contents. 

A large amount of syntonin and primary albumoses in the presence 



244 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

of a feeble peptone-reaction must, of course, be regarded as abnormal, 
pointing to a defective secretion of either hydrochloric acid or the 
enzymes, or of both. The same may be said to hold good when a 
pronounced peptone-reaction disappears upon the removal of syntonin 
and the primary albumoses. 

So far as the examination for the products of carbohydrate diges- 
tion is concerned, it may be stated, as a general rule, that in the 
presence of a normal amount of hydrochloric acid erythrodextrin can 
usually be demonstrated toward the end of gastric digestiou, while 
achroodextrin is nearly always obtained at that time when free hydro- 
chloric acid is absent, so that the tests for the presence of these two 
bodies may be regarded as roughly indicating the presence or absence 
of free hydrochloric acid. Boas draws attention to the fact, however, 
that ptyalin may, at times, though rarely, be absent, when conclusions 
drawn from these tests as to the presence of hydrochloric acid would 
be erroneous. 

The tests for sugar in the gastric juice do not furnish any infor- 
mation of practical value. 

Analysis of the Products of Albuminous Digestion. 

In order to separate the various bodies referred to from each other 
the following procedure may be employed : 

The filtered gastric contents are carefully neutralized with a dilute 
solution of sodium hydrate, using litmus-paper to determine the re- 
action ; a small drop of the mixture is placed upon the paper from 
time to time during the addition of the sodium hydrate until no 
change in color is produced either on the red or the blue paper. If 
syntonin is present, it will be precipitated, and can be collected on a 
small filter. Upon the addition of an excess of dilute acid or an 
alkali this precipitate will again be dissolved. The nitrate is feebly 
acidified by the addition of a few drops of a very dilute solution of 
acetic acid, treated with an equal volume of a saturated solution of 
common salt, and brought to the boiling-point. Any native albumin 
that may be present in solution is thus coagulated and can be filtered 
off on cooling. In the nitrate the albumoses and peptones remain. 
The presence of the former may be demonstrated by adding a few 
drops of nitric acid to a specimen, when a precipitate will form which 
dissolves upon the application of heat, and reappears on cooling; 
if necessary, the specimen may be diluted. 

Should the deutero-albumoses of vitellin or myosin be present, 
however, this test yields a negative result, and a precipitate only 
occurs when the solution, acidified with nitric or acetic acid, is com- 
pletely saturated with sodium chloride. 

The presence of primary albumoses may be established by adding 
pieces of rock-salt to the neutral solution, when a precipitate occurs. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 245 

The albumoses may roughly be separated from the peptones by satu- 
rating the acidified filtrate just obtained with pulverized ammonium 
sulphate, whereby the albumoses are precipitated almost entirely. 
A small portion of deutero-albuinoses, however, which resulted from 
the proto-albunioses, remains in solution and passes into the nitrate, 
which also contains all of the amphopeptone. In the nitrate this 
may be demonstrated as follows : a concentrated solution of sodium 
hydrate is added until all the ammonium sulphate has been trans- 
formed into sodium sulphate, and a slight excess of the hydrate is 
present ; care should be had, however, that the temperature does 
not rise too high, by immersion in cold water. The sodhmi sulphate, 
which separates out during this process, is allowed to settle. A 2 
per cent, solution of cupric sulphate is then carefully added drop 
by drop, to a specimen of the supernatant fluid, when in the pres- 
ence of peptones a rose to a purplish-red color will develop. 

To obtain the peptones, the filtrate is diluted with an equal volume 
of water, neutralized, and then treated with a solution of tannic acid, 
care being taken to avoid an excess, as otherwise the peptone precipi- 
tate is partly dissolved. 1 



Tests for the Products of Carbohydrate Digestion. 

Starch may be recognized by the fact that it strikes a blue color 
with a solution of iodo-potassic iodide, while the same solution gives 
a violet or mahogany brown with ervthrodextrin. To this end, it 
is only necessary to add a drop or two of Lugol's solution to a few 
cubic centimeters of the filtered gastric juice. The presence of 
achroodextrin may be inferred if no change in color occurs upon 
the addition of the reagent. 

Maltose and dextrose, which both react with Fehling's solution 
and undergo fermentation, differ from each other in the fact that 
the former does not reduce Barfoed's reagent on boiling. This is 
prepared by adding 1 per cent, of acetic acid to a 0.5 to 4 per cent, 
solution of cupric acetate. The rotatory power of maltose is about 
three times as strong as that of dextrose; (a) D = 150.4, as com- 
pared with 52.5. 

Lactic Acid. 

Mode of Formation and Clinical Significance. — It was for- 
merly thought that the acidity of the gastric juice was referable to 
the presence of lactic acid, as this can always be demonstrated in 
the beginning of the process of digestion. The hydrochloric acid 

1 For a more detailed account of the chemistry of digestion arid the analysis of the 
resulting products, see C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. 



246 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

was then supposed to result from the action of the lactic acid upon 
the chlorides of the food. That this view is erroneous C. Schmidt l 
succeeded in demonstrating beyond a doubt, as has been shown on 
page 217. An explanation of the presence of lactic acid suggested 
itself when Miller found that in the mouth various bacteria normally 
occur which are capable of forming lactic acid from sugar, and that 
from the gastric contents a number of bacteria can be isolated which 
are capable of causing acid fermentation in sugar-containing media. 
There would, hence, be nothing surprising in the constant occur- 
rence of lactic acid, as the two principal factors necessary for its 
formation are always present after the ingestion of an ordinary 
meal, viz., carbohydrates and bacteria capable of causing lactic acid 
fermentation. The absence of the lactic acid during the later stages 
of digestion was, furthermore, explained by the fact that lactic acid 
fermentation ceases in the presence of from 0.7 to 1.6 pro mille of 
hydrochloric acid — i. e., in the presence of amounts of hydrochloric 
acid which are found in the normal gastric juice. 

The normal occurrence of lactic acid in the stomach was, until 
recently, regarded as an established fact. But at this stage Martius 
and Liittke, employing the method already described, found " that the 
accurately determined curve of acidity referable to hydrochloric acid 
coincided in all respects, even at the beginning of the process of 
digestion, with the curve referable to the total acidity," so that 
lactic acid as a physiological constituent could not have been present. 
Recent researches of Boas, 2 moreover, appear to prove beyond a 
doubt that in physiological conditions no appreciable amounts of 
lactic acid are formed during the process of digestion, and that the 
lactic acid found after an ordinary meal has been introduced into 
the stomach as such. That lactic acid is actually present in the 
various kinds of bread has definitely been proved, and it is, hence, 
not permissible to make use of any test-meal containing lactic acid 
when the question as to its formation in the stomach is to be con- 
sidered. For these reasons Boas suggests the use of simple oatmeal - 
soup to which salt only has been added. For practical purposes 
this is probably not always necessary, as the small amount of lactic 
acid found after Ewald's test-breakfast may usually be disregarded ; 
an increased amount can be referred directly to pathological con- 
ditions. 

The fact that the lactic acid disappears, or is at least no longer 
demonstrable, at the height of digestion, Boas refers to a resorption 
or a carrying-off of the acid introduced, on the one hand, or to an 
interference of the hydrochloric acid with the delicacy of the reagent 

1 Loc. cit. 

2 J. Boas, "Ueber d. Vorkommen v. Milchsaure im gesunden u. krankeu Magen," 
Zeit. f. klin. Med., 1894, vol. xxv. p. 285. 



CHEMICAL EXAMINATION OF THE GASTUIC JUICE. 247 

usually employed — i. e., Uffelmann's reagent — on the other. Patho- 
logically the same rule may be said to hold good, as Boas was un- 
able to demonstrate its presence after the exhibition of his test-meal 
in the most diverse diseases of the stomach, viz., chronic gastritis, 
atony and dilatation referable to myasthenia, or pyloric stenosis 
following ulcer, etc. Mere traces, which were occasionally observed, 
are of no significance, and possibly referable to lactic acid fermenta- 
tion having taken place in the mouth. In all the cases examined, 
moreover, no organic acids could be demonstrated by the method of 
Hehner-Seemann (see page 255). 

It is apparent then that notwithstanding stagnation of the gastric 
contents and the absence of free hydrochloric acid in normal amounts, 
lactic acid is not necessarily formed in the stomach, even in the 
presence of carbohydrates. In only one disease of the stomach was 
lactic acid found in notable quantities, viz., in carcinoma. This ob- 
servation is in accord with the fact that UfFelmann's test here yields 
a marked reaction — i. e., a deep-lemon or a canary-yellow color — - 
even upon the addition of but a few drops of the gastric juice, while 
in the benign affections only a pale-yellow, brownish, or grayish 
color is obtained. 

Boas' test-meal should be given the evening before the examina- 
tion, the stomach having previously been washed free from all 
remnants of food ; the remaining contents are obtained the next 
morning. 

In an analysis of fourteen cases of carcinoma Boas was able to 
demonstrate the presence of lactic acid in amounts varying between 
1.22 and 3.82 pro mille in all cases but one, while in other diseases 
after the ingestion of Ewald's test-breakfast only 0.1 to 0.3 pro 
mille could be obtained. 

Unfortunately, recent investigations have shown that notable 
amounts of lactic acid may also be found in gastric anadeny, and in 
cases of dilatation referable to benign causes. Such cases, however, 
are rare, and it may safely be stated that the presence of large 
amounts of lactic acid will almost invariably justify the diagnosis of 
carcinoma of the stomach. 1 Lactic acid may, however, not be found 
in cases of pyloric carcinoma so long as any hydrochloric acid is 
secreted ; and may be present, on the other hand, in cases of stenos- 
ing gastritis referable to benign causes. 

That stagnation of the gastric contents and the absence of free 
hydrochloric acid alone are not capable of causing the formation of 
lactic acid has been seen, and it is, hence, difficult to explain why in 
carcinoma practically only lactic acid fermentation should occur. 

1 J.H. de Jong, "l?er Nachweis d. Milchsaure u. ihre klinische Bedeutung," Arch. 
f. Verdauungskrank., vol. ii. p. 53. J. Friedenwald, "' The Significance of the Presence 
of Lactic Acid in the Stomach." N. Y. Med. Jour., 1895. Eosenhaim u. Eichter, " Ueher 
Milchsaurebildung im Magen," Zeit. f. klin. Med., vol. xxviii. p. 505. 



248 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

Whether the malignant growth itself must be regarded as one of the 
principal factors in this connection, as Boas suggests, must still re- 
main an open question. 

Owing to the interest which attaches to this subject, it may not 
be out of place to refer briefly to the following observation of Koch : 
In a case in which ulcer of the stomach existed, the hydrochloric 
acid suddenly disappeared and gave place to lactic acid, which then 
steadily increased in amount from week to week. A tumor could 
not be demonstrated on physical examination. Soon after, the patient 
died, and at the autopsy a carcinoma of the stomach was found upon 
the base of the pyloric ulcer. An exploratory operation should hence 
be made whenever notable amounts of lactic acid can repeatedly be de- 
monstrated in the stomach contents after the ingestion of Boas' test-meal. 
Negative results, however, do not exclude the existence of carcinoma. 

The formation of lactic acid from starch may be represented by 
the following equations : 

(1) 2C 6 H 10 O 5 + H 2 = C 12 H 22 O n (milk-sugar). 

(2) C 12 H 22 O n + H 2 = 2C 6 H 12 6 (glucose). 

(3) 2C 6 H 12 6 = 4C 3 H 6 3 (lactic acid). 

It should, finally, be mentioned that only that form of lactic acid 
which results from fermentative processes is of interest in this con- 
nection, and not the sarcolactic acid contained in meat. 

Tests for Lactic Acid. — For the reasons indicated, Boas' test- 
meal (see page 212) should be employed whenever it is desired to test 
for lactic acid in the gastric contents. If the case under examina- 
tion shows well-marked symptoms of stagnation, the stomach should 
be washed out completely in the evening, the soup then given, and 
the gastric contents procured the next morning, before any food or 
liquid is taken. Otherwise the test-meal may be given in the morn- 
ing on an empty stomach, without previous lavage, and the contents 
examined one hour later. 

Uffelmann's Test. 1 — Heretofore Uffelinann's reagent was quite com- 
monly employed in testing for lactic acid, but everyone who has 
had occasion to make frequent use of this reagent in clinical work 
must have been struck with the uncertainty of the results so often 
obtained. In a large majority of the cases thus examined, particu- 
larly if Ewald's test-breakfast is employed, a characteristic reaction 
— i. e. y the occurrence of a lemon or canary-yellow color — is not 
seen, notwithstanding the presence of lactic acid, but a pale-yellow, 
brownish, grayish-white, or even gray color is obtained instead, often 
leaving in doubt whether lactic acid is present or not. Aside from 
doubtful results, the value of the test is greatly diminished by the 

1 Uffelmann, Deutsch. Arch. f. klin. Med., 1880, vol. xxvi.; and Zeit. f. klin. Med., 
vol. viii. p. 392. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 249 

fact that glucose, acid phosphates, butyric acid, aud alcohol give the 
same reaction, aud that iu the preseuce of such amounts of hydro- 
chloric acid as are found at the height of normal digestion lactic 
acid is not indicated by the reagent. All these difficulties have long 
been appreciated, and in order to obviate at least some of them it 
was proposed to apply the test to an aqueous solution of the ethereal 
extract of the gastric contents : 

To this end, 5 or 10 c.c. of the filtered gastric juice are extracted 
by shaking with from 50 to 100 c.c. of neutral sulphuric ether in a 
stoppered separating-funnel for about twenty or thirty minutes ; the 
ethereal extract is then evaporated on a water-bath or the ether 
distilled off (no flame). The residue is taken up with from 5 to 
10 c.c. of distilled water, and tested as follows : three drops of a 
saturated aqueous solution of ferric chloride are mixed with three 
drops of a concentrated solution of pure carbolic acid and diluted 
with water until an amethyst-blue color is obtained ; to this solution 
a portion of the ethereal extract is added, when in the preseuce of 
only 0.1 per cent, of lactic acid a lemon or canary -yellow color is 
obtained. 

Kelling's Method. 1 — Five or 10 c.c. of gastric juice are diluted 
with from ten to twenty volumes of water and treated with one or 
two drops of a 5 per cent, aqueous solution of ferric chloride. In 
the presence of lactic acid a distinct greenish-yellow color is seen if 
the tube is held to the light. This test is more reliable than that 
of Uffelmann, as a positive reaction is obtained only in the presence 
of lactic acid. 

Strauss' Method. 2 — Instead of evaporating the ether as in the 
above method, the ethereal extract may be directly examined by 
shaking with a freshly prepared solution of ferric chloride, as sug- 
gested by Fleischer. Making use of this principle, Strauss has 
constructed an apparatus (Fig. 46) which may be found very con- 
venient, and which permits of roughly determining the amount of 
lactic acid present. The instrument is essentially a separating- 
funnel of 30 c.c. capacity, bearing two marks, of which the one 
corresponds to. 5 c.c, the other to 25 c.c. The apparatus is filled 
with gastric juice to the mark 5, when ether is added to the 25 c.c. 
line. After shaking thoroughly, the separated liquids are allowed 
to escape by opening the stopcock until the 5 c.c. mark is reached. 
Distilled water is then added to the 25 mark, and the mixture treated 
with two drops of the officinal tincture of ferric chloride, diluted in 
the proportion of 1 : 10. Upon shaking, the water will assume an 
intensely green color if more than 1 pro mille of lactic acid is pres- 
ent, while a pale green is obtained in the presence of from 0.5 to 1 

1 G. Kelling, " Bhodan im Mageninhalt ; Zugleich ein Beitrag z. TTffelrnann'schen 
Milchsaurereagens," Zeit. f. physiol. Chem., vol. xviii. 

2 H. Strauss, "TJeber eine Modifikation d. Uffelmann'schen Reaktion," Berlin, klin. 
Woch.. 1895, No. 37. 



250 



THE GASTRIC JUICE AND GASTRIC CONTENTS. 



Fig. 46. 



pro mille. The tincture of iron should be kept in a dark-colored 
dropping-bottle of about 50 c.c. capacity. 

It will be observed that only large amounts of lactic acid, which 
alone are of importance from a diagnostic point of view, are indi- 
cated by the apparatus. Small amounts, as those introduced with 
Ewald's test-breakfast, or referable to lactic acid 
fermentation in the mouth, are not indicated, so 
that confusion as to the presence or absence of 
the acid can never arise. 

Boas' Method. 1 — In doubtful cases the follow- 
ing method should be employed, as with it, and 
following the exhibition of Boas' test-meal, all 
possible errors can be avoided. The stomach musty 
however, be washed perfectly clean before the test- 
meal is introduced. It is my belief that some of 
the positive results which have been obtained in 
other diseases than carcinoma are referable to 
neglect in this particular point. Aldehyde is not 
infrequently found in the stomach contents when 
sarcinse are present in large numbers, and may 
be mistaken for lactic acid, as I discovered to my 
regret not long ago. 

Principle of the Method. — When a solution of 
lactic acid is treated with a strong oxidizing agent 
and heated, the lactic acid is decomposed into 
acetic aldehyde and formic acid, according to the 
equation 



CH S — CH(OH)— CO.OH = CH 3 .CHO + H.CO.OH. 
Lactic acid. Acetic aldehyde. Formic acid. 



. _ Practically, then, the test for lactic acid resolves 

Strauss' apparatus for . _ . J ' 7 . 

the approximative itself into a test for acetic aldehyde, which can 

estimation of lactic -,.-, , • -i 1 , ■ • •<-! 

acid. readily be recognized by testing with various re- 

agents, such as an alkaline solution of iodo-potassic 
iodide, Nessler's reagent, and others. Nessler's reagent is prepared 
as follows : 2 grammes of potassium iodide are dissolved in 50 c.c. 
of water and treated with mercuric iodide while heating, until some 
of the latter remains undissolved. Upon cooling, the solution is 
diluted with 20 c.c. of water. Two parts of this solution are then 
treated with 3 parts of a concentrated solution of potassium hydrate ; 
any precipitate that may have formed is filtered off, and the reagent 
kept in a well-stoppered bottle. When aldehyde is added to such a 
solution a yellowish-red or red precipitate results, the exact color 
depending upon the amount of aldehyde present. One part of the 

1 Boas, Deutsch. med. Woch., 1893, No. 39; and Munch, med. Woch., 1893, No. 43. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 251 

aldehyde may still be recognized when diluted with 40,000 parts of 
water. 

With an alkaline solution of iodo-potassic iodide, aldehyde in a 
dilution of 1 : 20,000 will still produce a cloudiness, referable to the 
formation of iodoform, which is readily recognized by its character- 
istic odor (Lieben's test for acetone). 

Method. — The filtered gastric juice is tested for the presence of 
free acids with Congo-red (see page 225) If present, from 10 to 20 
c.c. are evaporated to a syrup on a water-bath, after the addition of 
an excess of barium carbonate, while the latter is unnecessary in the 
absence of free acids. The syrup is treated with a few drops of 
phosphoric acid, and the carbon dioxide removed by bringing it to 
the boiling-point once only, when it is allowed to cool and extracted 
with 100 c.c. of neutral sulphuric ether (free from alcohol), by shaking 
for half an hour. The layer of ether is poured off after half an hour, 
the ether is evaporated (no flame), the residue taken up with 45 c.c. of 
water, shaken and filtered, and finally treated with 5 c.c. of sulphuric 
acid and a pinch of manganese dioxide in an Erlenmeyer flask. 
This is closed with a perforated stopper carrying a glass tube bent 
at an obtuse angle, the longer limb of which passes into a narrow 
glass cylinder containing from 5 to 10 c.c. of Nessler's reagent or a 
like quantity of an alkaline solution of iodo-potassic iodide. If heat 
is now carefully applied, the aldehyde, formed by the oxidation of 
the lactic acid with manganese dioxide and sulphuric acid, passes 
over when the boiling-point is reached, and causes the precipitation 
of yellowish-red aldehyde of mercury in the tube containing the 
Nessler's reagent, or of iodoform if the alkaline solution of iodine 
is employed. 

Quantitative Estimation of Lactic Acid according to Boas' 
Method. 1 — The principle already set forth also applies to the quanti- 
tative estimation of lactic acid. 

Solutions required : 

1= A one-tenth normal solution of iodine. 

2. A one-tenth normal solution of sodium thiosulphate. 

3. Hydrochloric acid (sp. gr. 1.018). 

4. A potassium hydrate solution (56 : 1000). 

5. Starch solution. 
Preparation of these solutions : 

1. A normal solution of iodine should contain 126.53 (molecular 
weight of iodine) grammes of iodine in the liter, and a one-tenth 
normal solution, hence 12.6 grammes. In order to dissolve the 
iodine 25 grammes of potassium iodide are dissolved in about 200 
c.c. of distilled water, when the 12.6 grammes of resublimed iodine 
are added. This solution is then diluted with distilled water to the 
1000 c.c. mark, and requires no further correction. 

Loc. cit., p. 187. 



252 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

2. The one-tenth normal solution of sodium thiosulphate is pre- 
pared as described in the chapter on Acetone (see Urine). When 
treated with 1 gramme of ammonium carbonate pro liter it will 
retain its titre almost indefinitely. 

3. Preparation of the starch solution : 5 grammes of starch are 
dissolved in 900 c.c. of water by heating, wheD 10 grammes of zinc 
chloride in 100 c.c. of water are added. 

Method. — Ten to 20 c.c. of the filtered gastric juice are first 
treated as indicated above, viz., evaporated to a syrup after the 
addition of barium carbonate if free acids are present. A few 
drops of phosphoric acid are added, the carbon dioxide driven off 
by boiling, and the residue extracted, on cooling, with 100 c.c. of 
ether free from alcohol ; the ether is evaporated after separation, 
the residue taken up with 45 c.c. of distilled water, and treated 
with manganese dioxide and sulphuric acid. The flask is closed by 
a doubly perforated stopper ; through one aperture a bent tube passes 
to the distilling-apparatus, and a straight tube provided with a piece 
of rubber tubing, clamped off, through the other. The latter should 
dip well down into the liquid, and serves for passing a current of air 
through the solution when the distillation is completed. The mixt- 
ure is distilled until about four-fifths of the contents have passed 
over, excessive heat being carefully avoided, as otherwise the aldehyde 
will be decomposed, according to the equations : 

(1) CH 3 - CH(OH) - CO.OH = CH 3 .CHO + HCOOH. 
Lactic acid. Aldehyde. Formic acid. 

(2) CH3.CHO + HCOOH + 20 = CH 3 .COOH + C0 2 + H 2 0. 
Aldehyde. Formic acid. Acetic acid. 

To the distillate, which is best received in a high Erlenmeyer 
flask, well stoppered, 20 c.c. of the one-tenth normal solution of 
iodine are added, mixed with 20 c.c. of the 5.6 per cent, solution of 
potassium hydrate. The mixture is shaken thoroughly and allowed 
to stand for a few minutes. In order to liberate the iodine not used 
in the reaction, 20 c.c. of hydrochloric acid are added, and the ex- 
cess of iodine determined by titration with the one-tenth normal solu- 
tion of sodium thiosulphate. The titration is carried almost to the 
point of decolorization, when a little starch solution is added; the 
mixture is then titrated until the blue color has disappeared. The 
number of cubic centimeters of the one-tenth normal solution em- 
ployed, viz., 20, minus the number of cubic centimeters of the one- 
tenth normal solution of sodium thiosulphate, will then indicate the 
number of cubic centimeters of the former required for the formation 
of iodoform, viz., the amount of lactic acid present in 10 or 20 c.c. 
of gastric juice, as the case may be. As 1 c.c. of the one-tenth 
normal solution of iodine has been found to indicate the presence of 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 253 

0.003388 gramme of lactic acid, it is only necessary to multiply the 
number of cubic centimeters used by this figure, and the result by 
10, in order to obtain the percentage. 

The method described is reliable and sufficiently accurate for clini- 
cal purposes. At the same time it may be said that no more time 
is required than in the ordinary quantitative estimation of sugar by 
means of Fehling's method, or of hydrochloric acid according to 
the method of Martins and Luttke. 

Boas' Rapid Method. — This method is less accurate than the 
preceding one, but may be advantageously employed in the absence 
of the various reagents necessary with the former. Ten c.c. of 
filtered gastric juice are treated with a few drops of dilute sulphuric 
acid, and the albumin present removed by heat. The filtrate is evapo- 
rated to a syrup on a water-bath, water added to the original 
amount, and this again evaporated to a small volume, fatty acids 
being thereby removed. The lactic acid remaining is now extracted 
with ether (200 c.c. for every 10 c.c. of gastric juice) ; the ether is 
evaporated, the residue taken up with water and titrated with a 
one-tenth normal solution of sodium hydrate, using phenolphthalein 
as an indicator. As 40 parts by weight of sodium hydrate (molecu- 
lar weight) combine w T ith 90 parts by weight of lactic acid (molecu- 
lar weight), and as 1 c.c. of the one-tenth normal solution of 
sodium hydrate contains 0.004 gramme of sodium hydrate, the 
corresponding amount of lactic acid is found from the equation : 
40 : 90 : : 0.004 : x ; 40 x = 0.360 ; x = 0.009. The value of 1 c.c. 
of the one-tenth normal solution in terms of lactic acid is thus 0.009. 
By multiplying the number of cubic centimeters used by this figure, 
the amount of lactic acid present in 10 c.c. of gastric juice is 
ascertained. The result multiplied by 10 indicates the percentage. 

The Fatty Acids. 

Mode of Formation and Clinical Significance. — Unless much 
milk or carbohydrates have been ingested, fatty acids do not occur 
in the gastric contents under physiological conditions, and it would 
appear from the researches of Boas l that their formation is intimately 
associated with that of lactic acid. After the exhibition of his test- 
meal (see page 212) he was unable to demonstrate their presence 
either in health or in various diseases of the stomach, such as chronic 
gastritis, atony or dilatation referable to benign causes, etc. In 
carcinoma, however, fatty acids, just as lactic acid, were quite 
constantly found. 

That butyric acid can be derived from lactic acid has been demon- 
strated by Flugge, the reaction taking place according to the equation 

4H. 
1 Loc. cit. 



254 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

This observation is probably explained by the fact that most of the 
organisms causing butyric acid fermentation are anaerobic, while the 
Bacillus acidi lactici and the Oidium lactis eagerly absorb oxygen. 

Acetic acid fermentation, on the other hand, presupposes the pres- 
ence of alcohol, whether this is introduced into the stomach as such 
or whether it results from the action of yeast (Saccharomyces cere- 
visiaa) upon sugar. The transformation of alcohol into acetic acid 
is represented by the equation 

C 2 H 5 OH + 20 = C 2 H 4 2 + H 2 0, 

while the formation of alcohol during the process of fermentation 
from glucose is shown below : 

C 6 H 12 6 + 2H 2 == 2C 2 H 6 + 2H 2 + 2C0 2 . 

It is, hence, necessary, whenever acetic acid is met with in the 
gastric contents, to exclude the presence of alcohol, as only then 
is it permissible to refer its presence to stagnation and advanced 
decomposition of carbohydrates. 

If the examination is confined to an analysis of the gastric 
contents obtained otherwise than after the exhibition of Boas' or 
Ewald's test-meal, the diagnosis of pyloric stenosis with dilatation 
is probably always justifiable in the presence of notable quantities 
of butyric acid and acetic acid, while the same after a previous 
washing-out of the stomach and the exhibition of Boas' test-meal 
would suggest carcinoma as the cause of the stenosis. 

That butyric acid may occur in the gastric contents when butter 
or fats in general have been ingested is, of course, not surprising, 
and its presence then should be looked upon as a physiological occur- 
rence. At the same time it should not be forgotten that butyric acid, 
just as lactic acid, may possibly have been formed in the mouth, and 
conclusions should, hence, only be drawn when such sources of error 
can be definitely excluded, and the amount found exceeds mere traces. 

In conclusion, it may be said that in disease butyric acid is far 
more frequently encountered in the gastric contents than acetic acid, 
but the significance of the two, if alcoholism can be excluded, is the 
same. 

Tests for Butyric Acid. — 1. Butyric acid can usually be recog- 
nized by its odor alone, which is that of rancid butter. Often, how- 
ever, it will be necessary to resort to more definite tests, such as the 
following : 

2. Ten c.c. of filtered gastric juice are extracted with 50 c.c. of 
ether. The ether is evaporated and the residue taken up with a few 
cubic centimeters of water. If a trace of calcium chloride in sub- 
stance is now added, the butyric acid will separate out in the form of 
oil-droplets, the nature of which is readily recognized by the pungent 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 255 

odor. If, instead of adding calcium chloride, a slight excess of 
baryta-water is used, strongly refractive rhombic plates or granular, 
wart-like masses of barium butyrate are obtained upon evaporation. 

3. Butyric acid may also be recognized by the peculiar odor of 
pineapple which develops when the dry residue of the ethereal 
solution is treated with a little sulphuric acid and alcohol. The 
reaction is due to the formation of butyl ethylate (Pineapple test). 

Tests for Acetic Acid. — 1. Like butyric acid, acetic acid can 
usually be recognized by its odor. 

2. Ten c.c. of filtered gastric juice are extracted with ether. The 
ether is evaporated, the residue dissolved in a few drops of water, 
and accurately neutralized with a dilute solution of sodium hydrate, 
sodium acetate being formed. If to this a drop or two of a very 
dilute solution of ferric chloride is added, a dark-red color results 
in the presence of acetic acid. With silver nitrate a precipitate is 
obtained which is soluble in hot water. 

Quantitative Estimation of the Fatty Acids. — Method of Cahn- 
Mehring, modified by McNaught. 1 — The total acidity is determined in 
10 c.c. of filtered gastric juice. Another 10 c.c. are evaporated 
to a syrup, diluted with water, and similarly titrated. The difference 
between the two results will indicate the amount of fatty acids 
present. 

Quantitative Estimation of the Organic Acids. — Method of 
Hehner-Seemann. 2 — This method is based upon the observation that 
if a certain amount of a one- tenth normal solution of sodium 
hydrate is added to organic acids and the mixture is evaporated and 
incinerated, the organic acids are decomposed, with the liberation of 
carbon dioxide, while their alkali is left behind in the form of a 
carbonate ; this is then determined by titration with a one-tenth 
normal solution of hydrochloric acid. The amount of physiologi- 
cally active hydrochloric acid can be estimated at the same time by 
deducting from the total acidity the acidity referable to organic acids. 

Method. — Ten or 20 c.c. of filtered gastric juice are titrated with 
a one-tenth normal solution of sodium hydrate, evaporated to dry- 
ness, and incinerated, the application of heat being discontinued as 
soon as the ash has ceased to burn with a luminous flame. The 
residue is taken up with water and titrated with a one-tenth normal 
solution of hydrochloric acid. This is prepared by diluting 146 
grammes of the concentrated acid (sp. gr. 1.14) with distilled water 
to about 900 c.c, when the solution is brought to the proper strength 
by comparing it with a one-tenth normal solution of sodium hydrate, 
according to directions given elsewhere. The number of cubic cen- 
timeters of the one-tenth normal solution of hydrochloric acid 

1 Cited by Boas, Diagnostik u. Tberapie d. Magenkrankbeiten, 2d ed., 1891, p. 140. 

2 Seemann, "Ueber d. Vorbandensein freier Salzsaure im Magen," Zeit. f. klin. 
Med., vol. v. p. 272. 



256 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

employed multiplied by 0.00365 will indicate the amount of fatty 
acids in the 10 c.c. of gastric juice, in terms of hydrochloric acid ; 
the percentage is ascertained by multiplying by 10 or 5, as the case 
may be. By deducting the number of cubic centimeters employed 
from that of the one-tenth normal solution of sodium hydrate, first 
used, the number of cubic centimeters of the latter required for the 
neutralization of the physiologically active hydrochloric acid is 
ascertained, and the amount determined by multiplying by 0.00365. 

Gases. 

The stomach always contains a certain quantity of gases which 
have partly been swallowed and partly have passed into the stomach 
from the duodenum. As fermentative processes in health occur only 
when carbohydrates or fats have been ingested, and then only to a 
slight degree, nitrogen, oxygen, and carbon dioxide are the only 
gases found during the process of albuminous digestion. As the 
oxygen swallowed is, moreover, largely absorbed by the blood, and 
two volumes of carbon dioxide are returned for one volume of oxy- 
gen, the presence of large amounts of the former and small amounts 
of the latter is readily explained. In an analysis of the gases con- 
tained in the stomach of a dog which had been fed en meat, Planer 
found the following proportions : 

Carbon dioxide 25.2 vol. per cent. 

Oxygen 6.1 " " 

Nitrogen 68.7 " " 

"With a strict vegetable diet, on the other hand, hydrogen may 
also be found (Planer) : 

Man. Dog. 

Carbon dioxide .... 20.79 33.83 32.9 vol. per cent. 

Oxygen 0.37 0.8 " 

Nitrogen 72.50 38.22 66.3 " 

Hydrogen 6.71 27.58 

The presence of hydrogen is readily understood, if it is remembered 
that during the process of butyric acid fermentation hydrogen and 
carbon dioxide are formed. Lactic acid or acetic acid fermentation 
does not give rise to the formation of gases. 

Marsh gas, CH 4 , a product of the fermentation of cellulose, may 
also be found in pathological conditions, and is formed according to 
the equation 

(C 6 H 10 O 5 ) w + (H 2 0) w = 3(C0 2 )„ + 3(CH 4 ) W . 

It is yet an open question whether marsh gas is formed in the 
stomach or passes into the stomach from the small intestine. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 257 

Such observations must, however, be regarded as rarities. In 
one case of this kind, examined by Ewald and Ruppstein, 1 in which 
alcohol, acetic acid, lactic acid, and butyric acid were found in the 
vomited material, an analysis of the gases gave the following result : 

Carbon dioxide , 20.6 vol. per cent. 

Oxygen 6.5 " *' 

Nitrogen 41.4 " " 

Hydrogen 20.6 " " 

Marsh gas 10.8 " " 

Traces of olefiant gas and of hydrogen sulphide were also found. 
It is curious to note that in this case the patient, who, according to 
his own statement, had a " vinegar-factory in his stomach on one 
day and gas-works on another day," was occasionally able to light 
the eructated gas at the end of a cigar-holder, where it burnt with 
a faintly luminous flame. McNaught has reported a similar case, 
in which the analysis furnished the following results : carbon 
dioxide, 56 per cent.; hydrogen, 28 per cent.; marsh gas, 6.8 per 
cent.; atmospheric air, 9.2 per cent. 2 

Ammonia and hydrogen sulphide are also at times met with ; 
their presence is always due to albuminous putrefaction. 

Boas 3 found that hydrogen sulphide is quite commonly present 
in cases of dilatation referable to benign causes, while it is almost 
always absent in carcinoma. He adds that it is never found when 
lactic acid is present. In acute gastritis it may be observed tem- 
porarily. In a number of cases of carcinoma I have never found 
hydrogen sulphide. In one case reported by Strauss the Bacillus 
coli communis was apparently concerned in its production. 

To obtain a knowledge of the gases formed in the stomach during 
the process of digestion it is only necessary to fill an ordinary 
Doremus ureometer, or an Einhorn saccharimeter, with the unfiltered 
gastric contents, and to keep it at a temperature of from 37° to 
40° C, when the evolution of gas can be followed closely and the 
necessary tests made. The presence of carbon dioxide is readily 
recognized by passing a small amount of sodium hydrate, in concen- 
trated solution or in substance, into the tube, after the evolution has 
entirely ceased, when the fluid will rise. If other gases are present 
at the same time, they will remain after the carbon dioxide has been 
absorbed. Hydrogen sulphide is readily recognized by its odor 
and by the fact that it will color a piece of filter-paper, moistened 

1 Ewald, Arch. f. Anat. u. Physiol., 1874, p. 217. 

2 Kuhn, " Ueber Hefegahrung und Bildung brennbarer Gase im menschlichen 
Magen," Zeit. f. klin. Med., vol. xxi. : and Deutsch. med. Woch., 1892, No. 49, and 
1893, No. 15. 

3 Boas, " Ueber Schwefelwasserstoff bildung in Magenkrankheiten," Centralbl. f. 
inn. Med., 1895, No. 3; Deutsch. med. Woch., 1892, No. 49. Zawadzki, "Schwefel- 
wasserstoff im erweiterten Magen," Centralbl, f. inn. Med., 1894, No. 50. Dauber, 
"Schwefelwasserstoff im Magen," Arch. f. Verdauungskrank., vol. iv. p. 4. 

17 



258 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

with a few drops of sodium hydrate and lead acetate a more or less 
pronounced brown or black. The test is conveniently made by 
filling a test-tube about half-full with the gastric contents and clos- 
ing it with a cork stopper to which a strip of lead-paper, prepared 
as indicated, is fastened. 

The eructation of gas formed in the stomach should not be con- 
founded with the so-called eructatio nervosa, in which no gas is either 
eructated, or air simply enters the oesophagus and is expelled again 
with a loud, explosive noise. This may frequently be observed in 
neurasthenic and hysterical individuals, and is to a greater or less 
degree under the control of the will. It is hardly likely, however* 
that the physician will be called upon in the laboratory to differen- 
tiate between this form and that of true ructus, caused by fermenta- 
tive processes taking place in the stomach. The gases brought up 
in the former condition are without odor or taste, and thus differ 
from those found in true dyspepsia. 

Acetone. 

The presence of acetone in the gastric contents in pathological 
conditions has repeatedly been observed, especially by v. Jaksch and 
Lorenz, 1 and it is curious to note that the latter was at times able to 
demonstrate larger quantities of the substance in the gastric con- 
tents than in the urine. 

In the chapter on Acetonuria the relation existing between diges- 
tive diseases and the elimination of acetone will be dealt with more 
fully, but it may here be mentioned that in the primary diseases of 
the gastro-intestinal tract acetone is met with quite constantly in 
the gastric contents, while it is observed but rarely in the secondary 
forms, and never is seen in the gastric neuroses. This statement, 
however, is denied by Sovelieff, who claims to have found traces of 
acetone in only one case of nervous dyspepsia, while negative results 
were obtained in all other diseases of the stomach. I have re- 
peatedly been able to demonstrate the presence of acetone in cases 
of carcinoma, and never have found it in neurotic conditions. 

In order to test for acetone, the gastric contents are distilled after 
the previous addition of a small amount of phosphoric acid (1 : 
1000), when the tests of Reynolds and Gunning (see Urine) are 
applied to the distillate. If both reactions furnish a positive result, 
the presence of acetone may be regarded as demonstrated. Den- 
niges' test may also be employed, and can be applied to the filtered 
contents directly (see Urine). 

Ptomains and Toxalbumins. 

Remembering that ptomains and toxalbumins have been obtained 
directly from tainted meat, sausage, fish, clams, crabs, cheese, etc., it 
1 Lorenz, Zeit. f. klin. Med., 1891, vol. xix. p. 19. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 259 

is to be expected that these bodies may be met with in the gastric 
contents also. At the same time it may be mentioned that the 
stomach appears to possess the power of eliminating from the system 
poisons of this nature which are circulating in the blood. This is 
shown by the observations of Alt, who found that the water with 
which the stomach of an animal had been irrigated, after the sub- 
cutaneous injection of the poison of Pelias berus and Echidna 
arictans, or the direct bite of the snake, produced identical symp- 
toms of poisoning when injected into another animal. It is inter- 
esting to note that with lavage of the stomach the poisoned animal 
recovered. Similar observations have been made in cholera Asiatica. 
Certain vegetable alkaloids, such as morphin, are also known to be 
eliminated to a large extent by the stomach. Of the nature of the 
ptomai'ns and toxalbumins which may occur in the stomach, very 
little is known. 1 



Vomited Material. 

Food-material. — The vomiting of large amounts of totally undi- 
gested meat two or three hours after its ingestion is met with only 
in conditions associated with an entire absence of digestive juices 

Fig. 47. 




Collective view of vomited matter. (Eye-piece III., objective 8 A, Reichert.) a, muscle- 
fibres; b, white blood-corpuscles : c, & , squamous epithelium; c", columnar epithelium; d, 
starch-grains, mostly changed by the action of the digestive juices ; e, fat-globules ;/, sarcinse 
veutriculi ; g, yeast- fungi ; h, forms resembling the comma-bacillus found by the author once 
in the vomit of intestinal obstruction ; i, various micro-organisms, such as bacilli and micro- 
cocci ; k, fat-needles, between them connective-tissue derived from the food ; I, vegetable 
cells, (v. Jaksch.) 



from the stomach — i. e., in cases of atrophic cirrhosis of the stomach 
(anadeny of Ewald). This condition is not to be confounded with 

1 Brieger, Untersuchungen iiber Ptomaine, Hirschwald, Berlin, 1886. 



260 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

the regurgitation of undigested food, mixed with mucus and saliva, 
which is seen in cases of stricture of the oesophagus or of the car- 
diac orifice of the stomach. While at the outset of the latter 
disease the regurgitation of food occurs immediately, or at least 
very soon, after a meal, it may take place between meals in the 
later stages of the disease when dilatation has occurred. The 
recognition of the origin of the material brought up may then 
be exceedingly difficult. In such cases an examination should be 
made for biliary coloring-matter, which, if present, will, of course, 
immediately exclude the oesophagus as the source of the material 
ejected. Unfortunately, however,, the reverse does not hold good. 
Small amounts of undigested meat are of no significance. The 
vomiting of well-digested food is observed in some of the neuroses 
of the stomach, and also in certain cases of acute and subacute gas- 
tritis, ulcer of the stomach, and chronic gastritis in its early stages. 
The vomiting referable to cerebral and spinal diseases also belongs 
to this category. In this connection it is very important to inquire 
into the existence of nausea previous to the vomiting, for, as is well 
known, considerable amounts of saliva and mucus may be swal- 
lowed if much nausea has existed, the result being that the process 
of digestion is arrested before the occurrence of vomiting. In such 
an event it would be erroneous to conclude that, because the mate- 
rial ingested has not reached that stage of digestion which would be 
expected at the time of the vomiting, the stomach is incapable of 
properly performing its functions. 

Mucus. — The constant presence of large amounts of mucus in 
the gastric contents obtained with the stomach -tube is almost 
pathognomonic of the mucous form of gastritis, while its presence in 
vomited matter may be referable to pre-existing nausea. In cases of 
pharyngitis moderate amounts of mucus are frequently found. The 
vomiting of pure mucus, according to Boas, is always pathognomonic 
of the absence of dilatation of the stomach, a statement founded on 
reason, as it is altogether unlikely that no particles of food should 
be brought up at the same time. 

Under the term gastrosuceorrhoea mucosa Dauber l has described 
a condition in which large amounts of mucus are secreted by the 
non-digesting organ, in the absence of symptoms pointing to a 
gastritis. I have observed a similar case occurring in a neuras- 
thenic patient, in which enormous quantities of mucus could at times 
be obtained from the fasting organ, but never during the process of 
digestion. A mild degree of hyperchlorhydria existed at the same 
time, as well as enteritis mucosa and rhinitis mucosa. The motor 
power was practically normal. 

Mucus is readily recognized on simple inspection by its glossy 

1 Dauber, " Ueber kontinuirlicbe Magen-Schleimsecretion," Arcb. f. Verdauungs- 
krank., vol. ii. p. 167, 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 261 

appearance. Chemically, it is distinguished by its behavior toward 
acetic acid (see Urine). 

Saliva. — The vomiting of pure saliva in the morning upon rising 
is a fairly common symptom of chronic pharyngitis, which in turn 
frequently carries in its train a chronic gastritis ; it constitutes the 
so-called vomitus matutinus. Saliva, like mucus, is, of course, 
always present in the gastric contents in small amounts. Larger 
amounts are usually referable to an increased secretion owing to the 
existence of nausea. Chemically, saliva is best recognized by test- 
ing for the presence of the sulphocyanides (see Saliva, page 199). 

Bile. — Bile is rarely observed in the gastric contents brought up 
by the stomach-tube, but is frequently seen in vomited matter, of 
which it may be said to be a constant constituent whenever the 
vomiting has been very intense or frequently repeated. Its presence 
in the former case should always excite suspicion of the existence of 
stenosis of the descending or horizontal portion of the duodenum or 
the beginning of the jejunum. This diagnosis becomes the more 
probable the more constant its presence. 

Pancreatic Juice. — Mixed with the bile there is probably always 
present some pancreatic juice, and it has even been suggested that 
the constant absence of this constituent, in the presence of bile, is 
strongly suggestive of pancreatic disease or of obstruction of the 
pancreatic duct (the ductus Wirsungianus). 

Blood. — The presence of unaltered blood in the gastric contents 
is usually recognized without difficulty. As marked changes in 
color, varying from a deep red to a coffee or chocolate brown, may 
occur, however, when free acids are present, it is at times necessary 
to resort to a more detailed examination. In order to recognize mere 
traces when the macroscopical and even the microscopical examination 
do not point to the presence of blood, the method of Muller and 
Weber or that of Donogany should be employed. Kuttner claims 
that he was thus able to demonstrate the presence of blood in nu- 
merous cases of chlorosis in which other tests furnished negative 
results. I have been less successful in the disease in question, but 
admit that in cases of carcinoma and ulcer of the stomach it is with 
this method often possible to find traces of blood which would other- 
wise have remained unnoticed. 

Method of Muller and Weber. — The gastric contents are treated 
with a few cubic centimeters of strong acetic acid and extracted with 
ether. Should the ether not separate in a clear layer after a few 
minutes, a few drops of alcohol are added. If the ether then 
remains colorless, no blood-pigment is present, while a brownish- 
red color indicates the presence of acetate of hsematin. As a similar 
but yellowish-brown and much less intense discoloration of the 
ether may be produced by other pigments, such as biliary coloring- 
matter, it is well, in doubtful cases, to test the ethereal extract with 



262 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

tincture of guaiacum. A positive result indicates the presence of 
blood coloring-matter. The same may be said if, upon spectroscopic 
examination of the ethereal extract, an absorption-band is discov- 
ered at the junction of the red and yellow. 

Donogany's Method. — A small amount of the suspected material 
is extracted with a 20 per cent, solution of sodium hydrate and 
filtered. A drop of the filtrate is then mixed on a slide with a drop 
of pyridin and covered with a cover-glass, when, in the presence of 
blood, orange-red crystals of hsemochromogen will separate out on 
standing for a few hours. On spectroscopic examination these 
crystals will show the characteristic band of absorption between the 
yellow and the green. 

Hemorrhage from the stomach, hcematemesis, may be observed in 
the most diverse conditions. It is either dependent upon a primary 
disease of the organ, such as ulcer and carcinoma, or it occurs sec- 
ondarily to disease of other organs, leading to a hypersemic condi- 
tion of the gastric mucosa, such as the various forms of cardiac, 
renal, and hepatic disease, in connection with menstrual abnormali- 
ties, etc. In melsena, purpura hemorrhagica, pernicious ansemia, 
etc., the cause of the hemorrhage cannot always be determined. It 
appears to be certain, however, that nervous influences may also take 
part in the causation of gastric hemorrhage. 

Pus. — The occurrence of pus in vomited matter, referable to 
disease of the stomach itself, is uncommon. It is seen practically 
only in cases of phlegmonous and diphtheritic gastritis, and, as 
Strauss l has pointed out, in carcinoma affecting the smaller curva- 
ture and the region of the fundus. In such cases it is not uncom- 
mon to obtain as much as one-half to two tablespoonfuls of a 
mucopurulent fluid from the non-digesting organ. As the motor 
function in this form of carcinoma is often unimpaired, the symptom 
may be of considerable value in diagnosis. The presence of larger 
quantities usually indicates perforation into the stomach of an 
accumulation of pus from a neighboring organ. An abscess of the 
liver, a suppurative pancreatitis, an abscess of the colon, or a sub- 
phrenic abscess may thus prove to be its primary source. When 
present in considerable amount pus is, of course, readily detected 
with the naked eye ; if any doubt should arise, a microscopical 
examination will determine the question. 

Stercoraceous Material. — Very important from a clinical stand- 
point is the vomiting of stercoraceous matter which is notably 
observed in cases of ileus. Usually this is recognized without diffi- 
culty by its odor, which is referable to the presence of skatol. If 
any doubt should arise, it is only necessary to distil the vomited 
matter after the addition of a little phosphoric acid, and to test 
for the presence of phenol, indol, and skatol in the distillate, as 

1 H. Strauss, " Ueber Eiter im Magen," Berlin, klin. Woch., 1899, p. 870. 



CHEMICAL EXAMINATION OF THE GASTRIC JUICE. 263 

described in the chapter on Feces (see page 279). When chiefly 
derived from the small intestine, the vomited matter, according to 
v. Jaksch, will contain bile-acids and bile-pigment together with an 
abundance of fat, which may be detected by chemical or microscop- 
ical examination. The reaction is usually alkaline or feebly acid. 

I have had occasion to examine the vomited matter of a patient 
in whom an almost complete obstruction existed immediately above 
the ileo-caecal valve ; the color of the material was a golden yellow, 
the reaction neutral ; no bile-pigment or biliary acids were found, 
while hydrobilirubin was present. 

Parasites. — Of parasites, ascarides, segments of taeniae, trichinae, 
Anchylostoma duodenale, and Oxyuris vermicularis are, at times, 
encountered. Trichomonades has been described in the stomach 
contents of patients with carcinoma, by Hensen, Striibe, Zabel, 
Ullmann, and Cohnheim. The latter also refers to the occasional 
presence of amoebae and of megastoma, and is inclined to attach 
some diagnostic importance to the presence of such infusoria. He 
points out that the organisms in question will only find conditions 
favorable to their growth and development in the carcinomatous, 
non-insufficient organ ; while in non-malignant affections, as also 
in the malignant forms associated with insufficiency, the absence 
of an alkaline reaction and of irregularities in the surface of the 
mucosa (as in atrophy) prevent their active growth. Cohnheim 
regards their presence as important in the diagnosis of non-pyloric 
carcinoma, previous to the development of palpable masses, as the 
presence of lactic acid in the diagnosis of pyloric neoplasm. 

The examination for the presence of infusoria should be made at 
once after withdrawing the material from the stomach. The stom- 
ach contents should not be diluted. 1 For a description of these para- 
sites, see the chapter on the Feces. 

Odor. — The odor of normal gastric juice is peculiar, suggesting 
the presence of an acid, which can be sharply distinguished from 
acetic or butyric acid. If blood is present in large amount, the 
vomitus emits an odor which is perfectly characteristic. A feculent 
odor is met with in cases of enterostenosis or in the presence of an 
abnormal communication between the stomach and the small or 
large intestine. A putrid odor may be observed in cases of ulcera- 
tive carcinoma, pyloric stenosis referable to ulcer, simple carcinoma 
of the stomach, muscular hypertrophy of the pylorus, stenosis due 
to inflammatory adhesions, etc. In cases of phosphorus poisoning 
the vomited matter emits an odor of garlic ; the odor observed in 
uraemic conditions is referable to ammonia ; a carbolic acid odor is 
met with in cases of poisoning with this substance. 

1 G. Striibe, "Trichomonas hominis bei Carcinoma ventriculi," Berlin. klin. Woch., 
1898, p. 708. P. Cohnheim, Deutsch. med. Woch., 1903, vol. xxix. p. 206. 



264 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

MICROSCOPICAL EXAMINATION OF THE GASTRIC 
CONTENTS. 

In the gastric juice obtained from the non-digesting stomach the 
various morphological constituents of mucus and saliva, which have 
been described elsewhere, are found. Microscopical particles of 
food, such as elastic tissue-fibres, starch-granules, fat-droplets, fatty 
acid crystals, vegetable- and muscle-fibres, are, furthermore, quite 
constantly seen. Leucocytes and isolated nuclei also are observed ; 
the latter are set free by the action of the gastric juice upon the 
mucous corpuscles and epithelial cells. 

If gastric juice is allowed to stand, small tapioca-like bodies will 
collect at the bottom of the vessel, which upon microscopical exami- 
nation will be seen to contain numerous snail-shell-like formations, 
occurring either singly or collected in groups. These probably con- 
sist of altered mucin, as they can be produced artificially by adding 
a sufficient amount of dilute hydrochloric acid to saliva. Accord- 
ing to Boas, they are of no diagnostic significance. 

Epithelial cells, fragments of the epithelial lining of the ducts of 
glands, as well as goblet-cells, are not infrequently met with in the 
juice obtained from the non-digesting organ. In addition, various 
micro-organisms, such as the Leptothrix buccalis, Bacillus subtilis, 
saccharomyces, micrococci (often arranged in the form of tetrahedra), 
Clostridium butyricum, etc., may be encountered. 

Among the bacteria which may be found in the gastric contents 
under pathological conditions the bacillus described by Boas and 
Oppler l is undoubtedly the most important, and has of late attracted 
much attention. It appears to be present quite constantly in car- 
cinoma, and is almost always absent in other diseases of the stom- 
ach. It is thought that the formation of lactic acid, which is like- 
wise so constantly observed in carcinoma, is largely and perhaps solely 
referable to its presence. The organism in question (Plate XIV.) 
is non-motile, and essentially characterized by its great length 
and by the fact that the individual bacilli are frequently seen joined 
end to end, forming long threads and zigzag lines which are very 
characteristic. Often the entire field of vision is filled with dense 
conglomerations. Cultivation-experiments have thus far not been suc- 
cessful. The organism is readily stained with the usual anilin dyes. 
Tubercle bacilli may be found in vomited matter in cases of 
phthisis, where the sputa have been swallowed. Tubercular ulcera- 
tion of the stomach is exceedingly rare. Simmonds reports that 
in 2000 autopsies of tubercular individuals the condition was noted 
only eight times. 

1 Oppler-Boas, " Zur Kenntniss des Mageninhalts bei Carcinoma ventriculi," 
Deutsch. med. Woch., 1895, No. 5. Kauffmann, " Ueber einen neuen Milchsaure- 
bacillus," etc., Wien. klin. Woch., 1895, No. 8. Schlesinger u. Kauffmann, Wien. klin. 
Eundscbau, 1895, No. 15. 



PLATE XIV. 




L. SCHMIDT. FEC. 



The Boas-Oppler Bacillus, Stained with Methylene Blue. From a d 
of Carcinoma of the Large Curvature of the Stomach. 
(Personal Observation.) 



MICROSCOPICAL EXAMINATION OF THE GASTRIC JUICE. 265 

Sarcince (Fig. 47) occur in the form of peculiar colonies of cocci, 
arranged in squares or tetrahedra, strongly resembling cotton-bales. 
Not infrequently they are encountered under normal conditions, but 
only in small numbers. In pathological conditions, on the other 
hand, a drop of the gastric contents may constitute an almost pure 
culture. A case is even on record in which the pylorus had become 
entirely occluded by an inspissated mass of these organisms. When- 
ever present the existence of certain fermentative processes may 
be inferred. 

It is curious to note that in advanced cases of carcinoma of the 
stomach sarcinse are practically never seen, although the conditions 
are apparently most favorable for their development. Oppler 1 was 
unable to find them twenty-four hours after their introduction in 

Fig. 48. 




Cancer-cells from the gastric contents. (Ewald.) 

large numbers and in pure culture. In cases of carcinoma of the 
curvatures and the walls, as also in advanced pyloric carcinoma, 
sarcinse were never found, while they may be present in incipient 
cases of pyloric carcinoma so long as hydrochloric acid is secreted. 

In vomited material containing biliary coloring-matter, leucin, 
tyrosin, and cholesterin are quite commonly observed, and may be 
recognized by the form of their crystals, as well as by their chem- 
ical reactions, which are described elsewhere. 

The occurrence of blood and pus in the gastric contents has been 
considered (see pages 261 and 262). 

It not infrequently happens that small shreds of mucous mem- 
brane are brought away by the stomach-tube, and in cases of chronic 
gastritis, hyperchlorhydria not dependent upon ulcer, and in some 
1 Oppler, Munch, med. Woch., 1894, No. 29. 



266 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

of the neuroses, this is indeed not at all uncommon. 1 Boas even 
suggests that in the neuroses, where fragments of mucous membrane 
are so readily detached, this may possibly be connected etiologically 
with the formation of ulcers, and there can be no doubt that the 
mere action of the abdominal muscles exerted during the process 
of defecation may be sufficient to detach such fragments. From 
the microscopical appearance of the particles the diagnosis between a 
gastric neurosis and one of the various forms of chronic gastritis 
may frequently be made, and the same may be said to hold good in 
the differential diagnosis between a true gastritis and a glandular 
insufficiency referable to passive congestion of the gastric mucosa. 
At times tumor particles also are found in the gastric contents. 2 In 
the accompanying illustration (Fig. 48) a specimen obtained from a 

Fig. 49. 




A fragment of mucous membrane derived from the stomach. (Ewald.) 

carcinomatous patient is represented, which is readily distinguished 
from similar fragments of mucous membrane (Fig. 49). 

EXAMINATION OF THE MOTOR POWER OF THE 

STOMACH. 

Under physiological conditions the stomach should contain but 
few particles of food, or none at all, six hours after the ingestion of 
RiegePs meal, or one and one-half to one and three-quarters hours 
after that of Ewald. A delay in the propulsion of the gastric contents 
may be referable to the existence of a simple atony or to dilatation 
of the stomach. According to Boas, an atony may usually be diag- 
nosed if, following the exhibition of a supper consisting of bread and 
butter, cold meat, and a large cupful of tea, the stomach is found 
empty in the morning, providing, of course, that symptoms exist 
which point to atony or dilatation. It should be remembered, however, 
that in cases of acute and subacute gastritis, in the absence of a more 

1 M. Einhorn, Med. Eecord, June 23, 1894 ; Berlin, klin. Woch., 1895, No. 20 ; Arch, 
f. Verdauungskrankheiten, vol. v. Heft 3. 

2 P. Cohnheim, " D. Bedeutung kleiner Schleimhautstiickchen f. d. Diagnostik d. 
Magenkrankheiten," Arch. f. Verdauungskrankheiten, 1896, vol. i. p. 274. 



EXAMINATION OF THE MOTOR POWER OF THE STOMACH. 267 

serious lesion, food may be found in the stomach twenty-four hours 
after its ingestion. A dilatation may, on the other hand, be diagnosed 
if the stomach under the same conditions contains a considerable 
amount of food. In such cases it happens that not only remnants 
of the test-supper, but remains of meals taken one, two, three, or 
even more days previously are found. The quantities, moreover, 
which may be obtained at the time of examination are often surpris- 
ingly great, and may amount to sixteen pounds or more. Portel 
cites the case of the Due de Chausnes, one of Paris' greatest gour- 
mands, whose stomach could hold 4.5 liters — i. e., 8 pints. 

The following methods may be employed for the purpose of testing 
the motor power of the stomach : 

Leube's Method. 1 — Six hours after the ingestion of Riegel's 
meal the stomach is washed out with about 1000 c.c. of water. In 
the presence of only slight traces of food the motor power may be 
regarded as normal. This method is undoubtedly the most conven- 
ient for practical purposes. 

The Salol Test of Ewald and Sievers. 2 — This test is based upon 
the observation that salol, a compound ether of salicylic acid, is de- 
composed into phenol and salicylic acid only in an alkaline medium. 
As the salicylic acid is eliminated in the urine as salicyluric acid, it 
is possible to determine the time of the passage of the salol from the 
stomach into the small intestine. 

A capsule containing 1 gramme of salol is given to the patient 
immediately after his breakfast or dinner, when separate portions of 
urine, passed one-half, one hour, two hours, and twenty-four hours 
later, are tested by adding a small amount of a solution of ferric 
chloride. In the presence of salicyluric acid a violet color results. 
Under normal conditions a positive reaction is obtained after from 
forty-five to seventy-five minutes. A further delay may usually be 
regarded as indicating the existence of motor insufficiency. If no 
result is obtained after twenty-four hours, a pyloric stenosis undoubt- 
edly exists. Under normal conditions, furthermore, it will be 
observed that the salol elimination is completed after twenty-four 
hours, while in cases of dilatation of the stomach a positive reaction 
may still be obtained after thirty hours. It is thus possible to dis- 
tinguish between dilatation and descent of the stomach. 

The test, while it is convenient and usually yields fair results, is 
not altogether reliable, as the decomposition of the salol may at 
times occur in the stomach, owing to the presence of alkaline mucus, 
or may be delayed in the intestines owing to the existence of acid 
fermentation, etc. 3 

1 Leube, Deutsch. Arch. f. klin. Med., vol. xxxiii. 

2 Ewald u. Sievers, Therap. Monats., August, 1887. 

3 Brunuer, Deutsch. med. Woch., 1889. Huber, Correspondenzbl. f. schweizer Aerzte, 
1890. 



268 THE GASTRIC JUICE AND GASTRIC CONTENTS. 

EXAMINATION OF THE RESORPTIVE POWER OF THE 

STOMACH. 

To this end, a capsule containing 0.2 gramme of potassium iodide 
is given to the patient shortly before a meal, and the saliva examined 
for the presence of potassium iodide at intervals of from two to three 
minutes l (see Saliva, page 202). Under normal conditions a violet 
color is obtained after from six and one-half to eleven minutes, and 
a bluish tint after from seven and one-half to fifteen minutes. In 
pathological conditions a delayed reaction is observed in almost all 
diseases of the stomach, and is especially marked in cases of dilata- 
tion and carcinoma, less so in chronic gastritis, and variable in ulcer. 

Absolute conclusions, however, cannot be drawn from results thus 
obtained, as a normal reaction-time has also been observed in cases 
of dilatation .and chronic gastritis. 

INDIRECT EXAMINATION OF THE GASTRIC JUICE. 

Giinzburg's Method. 2 — In those cases in which for any reason the 
introduction of the stomach-tube is contraindicated or impracticable 
the following method, suggested by Giinzburg, may be employed : 

A tablet of 0.2 to 0.3 gramme of potassium iodide is inserted into 
a piece of the thinnest possible, strongly vulcanized rubber-tubing, 
measuring about 2.5 cm. in length. The ends are folded as shown 

Fig. 50. 




A fibrin-potassium-iodide package of Giinzburg. 

in Fig. 50, and the little package tied with three threads of fibrin 
hardened in alcohol. Every package should be examined before 
use, by immersion in warm water for several hours, to determine its 
tightness, testing for the presence of potassium iodide by means of 
starch-paper and fuming nitric acid. One of these packages is 
swallowed by the patient three-quarters to one hour after an Ewald 
test-breakfast, and the saliva tested for potassium iodide at intervals 
of fifteen minutes, until a positive result is reached or until six hours 
have elapsed. It is unnecessary to wait longer than six hours. In 
the presence of free hydrochloric acid the threads of fibrin are dis- 
solved and the potassium iodide absorbed. Under normal conditions 
a positive reaction is obtained after from one to one and three-quar- 
ters hours, while anachlorhydria undoubtedly exists if no result is 
obtained within five or six hours. In cases of hypochlorhydria the 
reaction is delayed for more than two to three hours. Giinzburg 

1 Penzoldt, Berlin, klin. Woch., 1892. Faber, Inaug. Diss., Erlangen, 1882. 

2 Sahli, Klinische Untersuchungsmethoden, 1900, p. 399. 



INDIRECT EXAMINATION OF THE GASTRIC JUICE. 269 

further advises that the resorption-test with potassium iodide be 
also made, and that the reaction-time be deducted from that taken 
up in the elimination of the iodide contained in the package. Sev- 
eral tests, moreover, should be made in the same case. 

I have had occasion to experiment with packages obtained from 
Germany, and manufactured according to the directions of Giinz- 
burg. 1 In most of the packages the threads of fibrin had become 
brittle and were broken in transit. The results obtained with about 
twenty intact specimens, however, were entirely satisfactory, and it 
is to be regretted that the packages cannot be obtained in the 
American market. 

Similar packages have been constructed by Sahli. 

Reach has of late made use of barium iodate and the oxyiodate of 
bismuth for the same purpose, but without enclosing the substance 
in rubber. As hydrochloric acid only is capable of liberating the 
iodine from these bodies, they may be employed instead of the Glinz- 
burg packages. As a result of his examinations, he concludes that 
in the presence of hydrochloric acid iodine can thus be demonstrated 
in the saliva within eighty minutes. He finds, however, that at 
times the reaction occurs later than might have been supposed from 
the amount of hydrochloric acid found. 

Simon's Method. — Personal researches have led me to believe 
that a close relation exists between the elimination of indican in the 
urine and the amount of free hydrochloric acid in the gastric con- 
tents. 2 The results reached may be summarized as follows : 

1. Euchlorhydria is associated rarely with an increased elimina- 
tion of indican. 

2. In cases of simple neurotic hyperchlorhydria a subnormal or 
normal amount of indican is the rule. 

3. In cases of hyperchlorhydria associated with ulcer an increased 
indicanuria is observed quite constantly. 

4. Anachlorhydria referable to organic lesions of the stomach is 
associated almost invariably with a highly increased indicanuria. 

5. Hysterical anachlorhydria may be associated with the elimina- 
tion of a normal or increased amount of indican. 

6. In cases of hypochlorhydria increased indicanuria is the rule. 
Given as premises : 

1. That a resorption of decomposing pus is not taking place any- 
where within the body, as such a process in itself is capable of caus- 
ing an increased elimination of indican. 

2. That a stenosis of the small intestine or a high degree of gas- 
tric atony does not exist. 

3. A normal mixed diet, containing no excessive amounts of red 
meat (see Indicanuria). 

1 Gothe Apotheke, Frankfurt a. M. 

2 C. E. Simon, Am. Jour. Med. Sci., 1895, vol. ex. p. 481. 



CHAPTER IV. 

THE FECES. 

The feces constitute a mixture of indigestible and undigested 
particles of food, of unabsorbed secretions of the gastro-intestinal 
tract, and their decomposition -products together with intestinal 
mucus, epithelial cells, and bacteria. 

EXAMINATION OF NORMAL FECES. 

General Characteristics. 

Number of Stools. — The number of stools which may be passed 
in the twenty-four hours is subject to wide variation, even under 
physiological conditions, but is usually constant for one and the same 
individual. One or two stools pro die may be regarded as normal. 
Exceptions, however, are frequent. Persons are thus met with who 
have but one stool every two to four days, and cases are on record 
in which only one passage occurred every seven to fourteen days, 
the individuals evidently enjoying perfect health. On the other 
hand, the number of stools may be increased to three or four under 
strictly normal conditions. Hence the importance of accurately ascer- 
taining the habitual number of stools in every individual. It would 
thus be manifestly wrong to regard the passage of three stools daily 
as diarrhoea, or the passage of only one stool in forty-eight hours as 
constipation, if this number has been habitual throughout life. 

Whether or not it is permissible to regard as normal those rare 
instances in which only one stool occurs every two to six weeks, or 
even less frequently, is rather doubtful. 

Amount. — In those cases in which more than one or two stools 
occur in twenty-four hours it is well to ascertain the amount actually 
passed. The normal amount varies between 100 and 200 grammes. 1 
This quantity is increased by a diet rich in vegetable and starchy 
foods, and is diminished by one rich in animal proteids, so that 60 
and 270 grammes may be regarded as the extreme limits in health. 
Such amounts as 500 and 1000 grammes are certainly abnormal. 

iVoit, Zeit. f, Biol., vol. xxv. p. 264. 
270 



EXAMINATION OF NORMAL FECES. 271 

Average quantities for various ages are given in the following 
table, which is taken from Schmidt and Strassburger : 1 

Average amount of 
Age» Diet. feces in twenty-four 

hours. 

Child, 1 month old . . . . . Mother's milk 3.3 Grammes. 

" 2-3 months old . . '. . " " 6.5 " 

" 7 " "...-. Variable 15-56 " 

"9 " " . . . . Cows' milk with addi- 
tions 59.0 " 
-2 years old Mixed 77.0 " 



4 
6 
9 

11 



Adult 



101.0 
134.0 
117.0 
138.0 
131.0 



Consistence and Form. — The consistence of a stool depends 
essentially upon the amount of water present, and hence upon the 
nature of the food ingested, being softer with a purely vegetable 
diet (80—85 per cent, of water) than with a diet rich in animal proteids 
(60— 65 per cent.). With a mixed diet the amount of water corre- 
sponds to about 75 per cent. As a general rule, normal stools 
exhibit the characteristic cylindrical form and are fairly firm. Mushy 
stools, however, are also seen quite frequently, and round, scybalous 
masses, although far more common in constipation, may likewise be 
observed in health. 

Odor. — The repugnant odor of the feces is, to a large extent, due 
to the presence of indol and skatol ; hydrogen sulphide, methane, and 
traces of phosphin may add still further to their disagreeable odor. 

Color. — The color of the feces varies, according to the nature of 
the food ingested, from a light to almost a blackish brown, a firm 
stool being in general darker than a thin stool. A stool that has 
remained exposed to the air is also somewhat darker upon its outer 
surface than in its interior, owing to processes of oxidation. In 
nursing-infants, in consequence of the exclusive ingestion of milk, 
the color is light yellow. 

Under normal conditions the color is never due to native biliary 
coloring-matter, but is largely dependent upon the presence of uro- 
bilin (see page 283). It is, furthermore, influenced by the nature 
of the food, chlorophyll tending to produce a greenish color, starches 
a yellowish tinge. If much blood is present in the food, the feces 
may be almost black, owing to the formation of hsematin. Huckle- 
berries and red wine likewise produce a blackish color, chocolate 
and cocoa a gray ; preparations of iron, manganese, and bismuth 
color the feces dark brown or black, owing to the formation of sul- 
phides of these metals ; the green color of calomel stools was formerly 

1 A. Schmidt and J. Strassburger, Die Faeces d. Menschen, Berlin, 1961, A. 
Hirschwald. 



272 THE FECES. 

supposed to be due to the formation of a sulphide, but is more likely 
caused by the presence of biliverdin. Santonin, rheum ; and senna 
produce a yellow color. 

Macroscopical Constituents. 

Alimentary Detritus. — Upon further examination of the feces it 
is possible to find visible to the naked eye undigested particles of 
food, which are partly indigestible and partly digestible, such as 
stones of cherries, grape-seeds, woody vegetable fibre, the skins of 
berries, large pieces of connective tissue, undigested pieces of apple, 
pear, potato, grains of corn, etc. Such undigested food is found in 
abundance when insufficiently masticated or taken in excessive 
amounts. 

Flakes of casein, recognizable with the naked eye, are also fre- 
quently seen. Care should be taken not to confound these with 
particles of stool composed of fatty acid crystals. This mistake is 
often made, and can readily be avoided by a microscopical or chemical 
examination (see page 295). 

Foreign Bodies. — In children, the insane, in cases of hysteria, 
and even in people who are apparently possessed of their normal 
senses, the physician must be prepared to find at times all kinds of 
foreign bodies, such as pins, coins, buttons, false teeth, tooth-plates 
with ragged edges, and even dirk-knives, all of which have been 
known to pass through the alimentary canal with perfect safety. It 
must not be forgotten, however, that in certain cases of hysteria 
bodies may be shown by patients which they claim have passed by 
the rectum, but which have been wilfully added to the stools, such 
as snakes, frogs, etc. 

Microscopical Constituents. 

Constituents derived from Food. — Microscopically, indigestible 
and undigested constituents of food may be seen (Fig. 51), such as 
the framework of vegetable material, sometimes still containing 
starch-granules or remnants of chlorophyll ; muscle-fibres, usually 
colored yellow and more or less altered in structure. Elastic-tissue 
fibres are readily recognized by their double contour and bold out- 
lines. Connective-tissue fibres of the white fibrous variety can also 
generally be distinguished ; when present in large quantities, how- 
ever, they are usually indicative of some digestive derangement, un- 
less they are observed following the ingestion of a meal particularly 
rich in meat. Flakes of casein also are seen frequently. 

Muscle-fibres are found in every stool whenever meat has been 
eaten. Under normal conditions, however, they are not numerous, 
unless particularly large quantities have been ingested. Their ap- 



EXAMINATION OF NORMAL FECES. 273 

pearance under the microscope may vary considerably. On the 
one hand, fibres are met with which still retain their characteristic 

Fig. 51. 




Collective view of the feces : a, muscle-fibres ; b, starch-granules ; c, vegetable material 
potato-cells ; e, egg of Uncinaria 
crystals; h, Charcot-Leyden crystals. 



d, potato-cells; e, egg of Uncinaria duodenalis; /, calcium oxalate crystals; g, fatty acid 

-Le ' 



features ; others are split up either partially or entirely into the 
well-known disks ; but more common than both are more or less 
roundish, yellow, apparently homogeneous fragments, which at first 
sight do not resemble muscle-fibres in the least. Upon closer in- 
vestigation, however, their true nature will become apparent. It 
will then be seen that two of the sides in some portions at least are 
more or less parallel, and if the specimen is examined with an oil- 
immersion lens some traces of cross-striation can probably always 
be discovered. 

Isolated starch-granules are scarcely ever found under normal 
conditions, excepting in young children who have been fed with much 
starchy material. Starch-granules enclosed in vegetable cells are 
likewise not found as a general rule, but are more common than the 
isolated granules. The presence of either in large numbers is usually 
indicative of the existence of some pathological condition affecting 
the gastro-intestinal tract. Their presence is easily recognized by 
treating microscopical preparations with a solution of iodo-potassic 
iodide (LugoPs solution), when the granules or fragments will assume 
a blue color. 

The presence of fat in the feces is quite constant, even in health. 
It may occur in the form of needle-like crystals, as fat-droplets, or 

18 



274 



THE FECES. 



as polygonal masses which are highly refractive and often colored 
yellow or a yellowish red. Their true nature is easily recognized 
by adding a drop of concentrated sulphuric acid and heating, when 
they are transformed into the characteristic fat-droplets. 

Morphological Elements derived from the Alimentary Canal. 
— 1. Epithelial cells. Well-preserved cylindrical or goblet cells are 
only exceptionally found in the feces, while transition-forms from 
the normal cells to mere spindles, in which a nucleus can no longer 
be recognized, are observed quite constantly. These degenerative 
changes, according to Nothnagel, 1 are the result of an abstraction 
of water from the cells, which may alter their appearance to an 
extent that only the experienced eye is capable of recognizing their 
true character. Pavement epithelial cells, when present, are derived 
from the anal orifice. 

2. Leucocytes are almost always absent in normal stools or pres- 
ent only in very small numbers. 

3. Red blood-corpuscles in very small numbers are occasionally 
observed under apparently normal conditions, but are then of no 
significance. 

4. In every stool a large number of structureless granules may 
be seen, lying either by themselves or collected into heaps ; they are 
designated as detritus. 

Crystals. — Needle-like crystals of free fatty acids, and the cal- 

Fig. 52. 



fllPiiP 



Fatty crystals obtained from the feces. 



cium and magnesium salts of the higher members of this group, 
occurring either singly or arranged in sheaves, may be found in 
every stool (Fig. 52). They are of no significance unless present 

1 Nothnagel, Beitrage z. Physiol, u. Pathol, d. Darmes, Hirschwald, Berlin, 1884, 
and Specielle Pathol, u. Therap., Holder, Wien, 1895, vol. xvii. Pt. 1. 



EXAMINATION OF NORMAL FECES. 275 

in large numbers. Nothnagel 1 speaks of the frequent occur- 
rence of certain calcium salts (of fatty acids, as he believes) in 
normal as well as pathological stools. He states that they are 
almost always bile-stained, and occur in irregular, sometimes ellip- 
tical, oval, or circular masses, in which a crystalline structure 
cannot be distinguished. They are apparently of no importance. 
Quite common, also, are crystals of neutral calcium phosphate and 
ammonio-magnesium phosphate, the former occurring in the form 
of more or less well-defined wedge-shaped crystals collected into 
rosettes, the latter presenting the well-known coffin-shape when the 
stool is mushy, while in firm stools irregular fragments mostly are 
found. At one time the ammonio-magnesium phosphate crystals 
were supposed to be characteristic of typhoid stools, but it is now 
known that they occur in normal feces, as well as under the most 
varied pathological conditions. Their presence is of no diagnostic 
significance. It is important to note that the neutral phosphates 
are never stained by bile-pigment, and the triple phosphates only 
in rare instances. Both are easily soluble in acetic acid. Crystals 
of calcium oxalate may be found in abundance following the inges- 
tion of certain vegetables, such as sorrel and spinach. They are 
usually found imbedded in the vegetable debris. They are readily 
recognized by their characteristic envelope-form, their insolubility 
in acetic acid, and their solubility in hydrochloric acid. Not infre- 
quently they are bile-stained. 

Calcium lactate is frequently seen in the stools of children re- 
ceiving a milk-diet ; they occur in the form of sheaves composed of 
radiating needles. Calcium carbonate is rarely observed, but occa- 
sionally occurs in the form of amorphous granules or dumb-bell- 
shaped crystals. Calcium sulphate crystals are likewise rare, but may 
be produced artificially by the addition of sulphuric acid, when beauti- 
ful needles and platelets may be observed. Cholesterin, while always 
present in solution, is rarely observed in crystalline form (Fig. 53). 
I have found it only twice in several hundred examinations. Hsema- 
toidin crystals are never found in normal stools. Charcot-Leyden 
crystals may be found under pathological conditions ; according to 
my experience, they are never seen in normal stools. 

Parasites. — The parasites which occur in normal feces may be 
divided into vegetable and animal parasites. 

Vegetable Parasites. — These are always present in enormous 
numbers. What relation they bear to the process of digestion is 
still an open question. The idea held by Pasteur and many others, 
that animal life cannot go on in the absence of bacteria from the 
digestive tract has been disproved by Nuttall and Thierfelder. 2 A 

1 Loc. cit. 

2 Nuttall u. Thierfelder, " Thierisches Leben ohne Bakterien im Darm," Zeit. f. 
physiol. Chem., 1896, vol. xxi. p. 109, and 1897, vol. xxii. p. 62. 



276 THE FECES. 

guinea-pig, removed by Csesarean section from the uterus of the 
mother-animal, under antiseptic precautions, was placed in a ster- 
ilized glass cage and nourished for a week with sterilized food. The 
air which the animal breathed was likewise sterilized. During this 
week the animal consumed about 330 c.c. of milk and appeared to 
be normal in every respect. At the expiration of the week it was 
killed, when a microscopical examination of the intestinal contents 
revealed the absence of bacteria. Culture-experiments also were 
negative. 

Macfadyen, Nencki, and Sieber 1 likewise found that their now so 
often quoted fistula patient continued in good health, and even 
gained flesh, although the entire large intestine, in which bacterial 
activity is always greatest, was isolated for a period of many weeks. 

Fungi. — Fungi, with the exception, perhaps, of the Oi'dium albi- 
cans, which has at times been observed, are rarely found in the feces. 

Schizomycetes. — Saccharomyces cerevisise is one of the normal 
constituents of the feces, and is found in its characteristic forms, 
three or four buds, however, being but ordinarily observed. Owing 
to the glycogen present in their substance, they assume a mahogany 
color when treated with a solution of iodo-potassic iodide. They 
should not be confounded with a class of bacteria which closely re- 
semble the saccharomyces in general appearance, but are colored blue 
when treated in the same manner (see below). 

Bacteria. — The bacteria are the micro-organisms xav y i^o^/ju 
which are found in the feces. Their number is truly enormous. 
Sucksdorif thus found in his own person that on an average 53,124,- 
000,000 were eliminated in the twenty-four hours under normal 
conditions. About 97 per cent, of these are directly derived from 
the ingested food, and the remaining 3 per cent, from swallowed 
saliva. If we recall the strongly bactericidal power of the gastric 
juice, such an observation must at first sight appear most surprising. 
It should be remembered, however, that the spores of the bacteria 
are far less susceptible to the action of hydrochloric acid, and that 
large amounts of the ingesta are carried into the small intestine at 
a time already, when hydrochloric acid has not as yet appeared in 
the free state. 

On the whole, the bacteriological flora of the intestinal contents 
is fairly constant, but, as in the other cavities and channels of the 
body where bacteria are invariably met with, transient guests are 
also not uncommon. The majority of the bacteria which are here 
encountered are, as a general rule, harmless ; but it is important to 
note that under suitable conditions a number of these may develop 
pathogenic properties. Broadly speaking, the bacteria which may 

1 Macfadyen, Nertcki, u. Sieber, " Untersucbungen iiber die cbemischen Vorgange 
ini mengcblichen Dihmdarni," Arcb. f. exper. Path. u. PharmakoL, 1891, vol. xxviii. 
p. 311. 



EXAMINATION OF NORMAL FECES. 211 

be found normally in the feces can be divided into two classes. 
Those belonging to the first order are stained a yellow or a yel- 
lowish brown with iodo-potassic iodide, while those belonging to 
the second class are colored blue or violet by the same reagent. To 
the former belong the Bacterium termo, the Bacillus subtilis, and a 
large number of micrococci ; into a description of these it is not 
necessary to enter at this place. 1 Under the second heading v. Jaksch 2 
describes the following forms : 

1. Micrococci occurring in the zooglcea stage, which are colored 
a violet red. 

2. Short, thin rods, tapering slightly at both ends, and in their 
microscopical appearance much resembling the bacillus of the septi- 
caemia of mice ; sometimes they contain one or two little bodies, 
which are not stained by the reagent. 

3. Short or long rods, which resemble the Leptothrix buecalis in 
their behavior toward iodo-potassic iodide. 

4. Bacilli resembling the Bacillus subtilis. 

5. Bacillus butyricus. This micro-organism, according to Brieger, 
is the cause of butyric acid fermentation. It occurs in the form of 
broad rods with rounded extremities, but may also be elliptical or 
spindle-shaped. With Lugol's solution it is colored blue or violet, 
either entirely or only in its central portion. 

6. Large round forms, characterized, when unstained, by a pale 
lustre, and which very much resemble yeast-cells (see above). 

7. Micrococci, which assume a reddish, but not very pronounced 
tint. 

It should be mentioned that this second class of micro-organisms 
is not so largely represented in the feces as the first. 

To speak more specifically, the following bacteria have thus far 
been isolated from the feces : the Bacillus coli communis, Bacterium 
lactis aerogenes, Bacillus subtilis, Proteus vulgaris, Bacillus putrifi- 
cus coli, Bacillus liquefaciens ilei, Bacterium ilei, Bacterium ovale 
ilei, Bacillus gracilis ilei, the veil bacillus of Escherich, Bacillus 
butyricus, Bacillus Uptadel ; Streptococcus coli gracilis, Strepto- 
coccus coli brevis, Streptococcus liquefaciens ilei, Streptococcus pyo- 
genes duodenalis, Staphylococcus liquefaciens albus, Staphylococcus 
liquefaciens flavus, Micrococcus ovalis, the porcelain-coccus of 
Escherich, tetradenococcus. In addition, various other bacteria have 
been found, but have not as yet been obtained in pure culture. This 
is true more particularly of certain forms of spirillum. 

The specific pathogenic bacteria which may be found in the feces, 
as well as those above mentioned, which may at. times develop 
pathogenic properties, will be described in detail later on. 

Animal parasites are probably never present under strictly normal 
conditions. 

1 Fliigge, Die Microorganismen. 

2 v. Jaksch, Klinische Diagnostik, 1896. 



278 THE FECES. 

Chemistry of Normal Feces. 

Reaction. — The reaction of the feces is usually alkaline, sometimes 
neutral, rarely acid, the alkalinity being due to ammoniacal fermen- 
tation, the acidity to lactic and butyric acid fermentation, taking 
place in the intestines. In infants the stools are normally acid. 

General Composition. — The following table, taken from Gautier, 
will give an idea of the composition of fresh feces, calculated for 
1000 parts by weight : 

Adult man. Suckling. 

Water 733.00 851.3 

Solids . 267.00 148.7 

Total organic material 208.75 137.1 * 

Total mineral material 10.95 2 13.6 

Alimentary residue 83.00 

The organic material yielded : 

Aqueous extract 53.40 53.50 

Alcoholic extract .'.. 41.65 8.20 

Ethereal extract 30.70 17.60 3 

In addition, there are gases, which vary in quantity according to 
the nature of the food ingested, such articles as beans, heavy bread, 
potatoes, etc., increasing the amount very considerably. 

Milk diet. Meat diet. Vegetable diet. 
Per cent. Per cent. Per cent. 

Carbon dioxide 9-16 8-13 21-34 

Hydrogen 43-54 0.7-3 1.5-4 

Marsh gas 0.09 26-37 44-55 

Nitrogen 36-38 45-64 10-19 

Of these gases, carbon dioxide is partly referable to alcoholic and 
butyric acid fermentation, and partly to albuminous putrefaction, 
taking place in the intestines. Marsh gas is formed during the fer- 
mentation of cellulose, while the nitrogen has partly been swallowed 
and is partly referable to albuminous putrefaction. A portion also 
is probably derived from the blood, and it may be mentioned in this 
connection that the enormous quantities of carbon dioxide so often 
discharged in cases of hysteria are undoubtedly referable to this 
source, the gas passing from the blood through the gastro-intestinal 
mucous membrane into the stomach and intestines. 

In order to give a general idea of the chemical constituents of the 
feces these may be divided into : 

1. Food material which could be assimilated, but which was taken 
in excess, such as starches, fats, and a small amount of non-assimi- 
lated albuminous material. 

2. Indigestible substances, such as chlorophyll, gums, pectic 

1 Including 54 parts of mucin, epithelium, and calcareous salts. 

2 Not comprising earthy phosphates. 



Of this, 3.2 is cholesterin. 



EXAMINATION OF NORMAL FECES. 279 

products, resins, various coloring-matters, nucleins, chitin, and 
insoluble salts, viz., silicates, sulphates, earthy phosphates, ammonio- 
magnesium phosphate, etc. 

3. Products derived from the digestive canal, as mucus, partly 
transformed biliary acids, dyslysin, cholesterin, lecithin. 

4. Substances in process of absorption, as emulsified fats, fatty 
acids, leucin, and biliary acids. 

5. Products of decomposition, referable to microbic activity, such 
as fatty acids, comprising the entire series from acetic to palmitic 
acid, the latter being especially abundant ; lactic acid, phenol, cresol, 
indol, skatol, excretin, leucin and tyrosin ; phenol-propionic, phenyl- 
acetic, hydroparacumaric, and parahydroxyl-phenyl-acetic acids ; 
ammonium carbonate, and ammonium sulphide. 

6. Products of metabolism eliminated through the intestines : 
urea, uric acid, and xanthin-bases. 

7. Pigments : stercobilin, hsematin, hydrobilirubin, coloring-mat- 
ter derived from the blood, and, in abnormal conditions, bile-pig- 
ments. 

8. Water. 

9. Gases, as carbon dioxide, marsh gas, hydrogen, and nitrogen. 
The study of these substances as a whole, as well as in detail, 

is of great importance, not only from the standpoint of the physiolo- 
gist, but also from that of the clinician, giving, together with a 
careful urinary analysis, the clearest idea of the metabolic processes 
taking place in the body. 

The chemical study of the feces, however, has so far received but 
little attention, and data of practical importance have scarcely been 
obtained from the work accomplished. The field is nevertheless an 
important one. 

It is impossible to give here a detailed description of the various 
chemical constituents which have been mentioned. Only the most 
important ones, and those especially interesting from a physiological 
and pathological standpoint, will be considered. 

Phenol, Indol, and Skatol. — Phenol, indol, and skatol are 
formed during the process of albuminous putrefaction, and are con- 
stant constituents of the feces. A small portion is resorbed from 
the intestinal canal, and appears in the urine in combination with 
sulphuric acid and to a slight extent also with glucuronic acid. Pre- 
viously, however, the indol and skatol are oxidized to indoxyl and 
skatoxyl, respectively (see Urine). 

To demonstrate the presence of phenol, indol, and skatol in the 
feces, we may proceed as follows : 

The feces are diluted with water, acidified with phosphoric acid, 
and distilled. The volatile fatty acids present, together with phenol, 
indol, and skatol, pass over. The distillate is then neutralized with 
sodium carbonate and again distilled. During this process phenol, 



280 THE FECES. 

indol, and skatol pass over, the fatty acids remaining behind as 
sodium salts. In order to separate the phenol from indol and skatol, 
the distillate is alkalinized with potassium hydrate and again distilled. 
The phenol now remains behind, and may be obtained in pure form 
by distilling with sulphuric acid ; in this final distillate its presence 
may be demonstrated by the following reactions : 

1. With ferric chloride phenol yields an amethyst-blue color. 

2. With bromine-water a crystalline precipitate of tribromophenol 
is obtained. 

3. Treated with Millon\s reagent — i. e., the acid mercuric nitrate — 
a red color develops. 

Indol and skatol pass over after treating the above mixture of 
the three with potassium hydrate and distilling. These two bodies 
may then be separated from each other by taking advantage of their 
different degrees of solubility in water. 1 

Indol forms small plates, melting at 52° C, which are easily 
soluble in hot water, alcohol, and ether ; its odor is feculent. 

Reactions of indol : 1. When treated with nitric acid and a little 
sodium nitrite a crystalline red precipitate of the nitrate of nitroso- 
indol is obtained. 2. A small piece of pine wood moistened with 
an alcoholic solution of indol acidified with hydrochloric acid is 
colored a cherry red. 

Skatol crystallizes in plates which melt at 95° C. They are soluble 
with more difficulty in water than indol, and emit a feculent odor. 

Reactions of skatol : 1. With nitric acid and sodium nitrite only 
a milky cloudiness results. 2. Pure skatol does not color pine wood 
moistened with hydrochloric acid ; but if a bit of the wood is satu- 
rated with a dilute alcoholic solution of skatol and then immersed 
in strong hydrochloric acid, it assumes a cherry-red and later a 
bluish-violet color. 3. With nitric acid of a specific gravity of 1.2 
it gives a marked xanthoproteic reaction on boiling — i. e., a yellow 
color which turns to orange upon the addition of an excess of 
ammonia. 

The determination of cresol in the presence of phenol, together 
with which it is obtained, is, when only small quantities of these 
substances are present, a difficult matter. They may be separated 
from each other by transforming both into their sulpho-acids ; the 
barium salt of para-sulpho-phenol is practically insoluble in barium 
hydrate. 

Fatty Acids. — The chemical composition of fatty acids present 
in the feces, as well as the relation existing between them, Is shown 
in the table below. The formula C n H 2 „ +1 , COOH, or C n H 2n 2 ex- 
presses their general structure. 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. 



EXAMINATION OF NORMAL FECES. 281 

Formic acid . H.COOH = G H 2 2 

Acetic acid CH 3 .COOH = C 2 H 4 2 

Propionic acid CH 3 .CH 2 .COOH = C 2 H^ 2 

Butyric acid CH 3 .(CH 2 ) 2 .COOH = C 4 H 8 2 

Isobutyric acid (CH 3 ) 2 .CH.COOH =C 4 H 8 2 

Valerianic acid CH 3 :(CH 2 ) 3 .COOH =C 5 H lt ,0 2 

Caproicacid CH 3 .(CH 2 ) 4 .COOH =C 6 H 12 2 

Capric acid CH 3 .(CH 2 ) 8 .COOH = C^H^O, 

Palmitic acid CH 3 .(CH 2 ) u .COOH = C 16 H 32 2 

Stearic acid CH 3 .(CH 2 ) 16 .COOH — C 18 H 36 2 

These acids are derived partly from fats, partly from carbohydrates, 
and to some extent also from proteids. 

Separation of the Fatty Acids from the Feces. — If the distillate, neu- 
tralized with sodium carbonate, referred to in the above method, is 
again distilled, the sodium salts of the fatty acids remain behind : 

2C 15 H 31 .COOH -f Na 2 C0 3 =z 2C 15 H 31 .COONa + H 2 + C0 2 . 

The solution is then evaporated to dryness on a water-bath, the 
residue extracted with alcohol, the alcohol evaporated, and the final 
residue dissolved in water. This solution may now be further ex- 
amined. In order to separate the different fatty acids from each 
other, it is best, if the quantity is sufficiently large, to transform 
them into their silver or barium salts, and to separate these by their 
varying degrees of solubility in water or by fractional distillation. 

General properties of the fatty acids : they are all monobasic, 
and soluble in water, alcohol, and ether. Their alkaline salts are 
readily soluble in water and alcohol, but insoluble in ether. The 
silver salts are dissolved with difficulty. 

1. Formic acid is a colorless liquid, of a penetrating odor, boil- 
ing at 100° C. A concentrated solution of its alkaline salts is 
precipitated by silver nitrate ; the silver salt becomes black on 
standing, and reduction takes place at once upon the application of 
heat. Treated with ferric chloride in neutral solution it yields a 
blood-red color, which disappears on boiling, while a rust-colored 
precipitate is formed at the same time. 

2. Acetic acid is a liquid of a pungent odor, which boils at 
119° C. After neutralization a blood-red color is obtained on the 
addition of ferric chloride. Neutral solutions of its salts with the 
alkalies yield a precipitate with silver nitrate, which is soluble in 
hot water without reduction taking place. 

3. Propionic acid is an oily fluid, boiling at 117° C. With 
ferric chloride no red color results ; with silver nitrate it behaves 
like formic acid. 

4. Butyric acid is an oily liquid, boiling at 137° C. ; its odor is 
similar to that of rancid butter. When treated with an acid, its 
salts give off the characteristic odor ; with ferric chloride it yields 
no red color ; with silver nitrate its alkaline salts form a crystalline 
precipitate which is insoluble in cold water. 



282 THE FECES. 

5. Valerianic acid boils at 176.3° C, and has a penetrating, dis- 
agreeable odor. Its silver salt crystallizes in plates, which are 
soluble with difficulty. 

Cholesterin. — Cholesterin (C^H^O) occurs in small amounts in 
almost all animal fluids. It is found also in various tissues of the 
body, especially in the brain. Its origin and mode of formation in 
the various organs of the body, as well as the cause of its presence 
in the alimentary canal, are as yet unknown. It crystallizes in 
colorless, transparent plates, the margins and angles of which usually 
present a ragged appearance (Fig. 53). It is practically insoluble 
in water, dilute acids, and alkalies. In boiling alcohol it is readily 
soluble and crystallizes out from this solution on cooling; it is 
likewise easily soluble in ether, chloroform, and benzol. 

In order to obtain cholesterin from the feces, in which it is always 
present, though rarely in crystalline form, the fatty acids, phenols, 
indol, and skatol must first be distilled off, as described, when the 
residue is strongly acidified with sulphuric acid, extracted with 
alcohol, and then with ether. The ethereal extract is filtered, the 
ether distilled off, and the residue digested with sodium carbonate, 
in order to transform into their salts any fatty acids which may still 
be present. This mixture is then evaporated to dryness, and again 
extracted with ether. The alcoholic extract above mentioned is also 
filtered, supersaturated with sodium carbonate, the alcohol distilled 
off, the residue dissolved in water and likewise extracted with ether. 
In the watery alkaline residue there remain bile-acids, oleic, palmitic, 
and stearic acids, which can be separated by transforming them into 

Fig. 53. 







Cholesterin crystals. 



their barium salts. The cholesterin and fats pass over into the 
ether. This is distilled off and the residue treated with an alcoholic 
solution of potassium hydrate. The alcohol is evaporated on a 
water-bath, the remaining liquid diluted with water and again ex- 



EXAMINATION OF NORMAL FECES. 283 

tracted with ether. The fats remain in the aqueous solution as 
soaps, while the cholesterin has passed into the other. 

Tests for cholesterin : 1 . Under the microscope add a drop of 
concentrated sulphuric acid to some of the crystals ; they gradually 
disappear, the edges assuming a yellowish-red color. 

2. Dissolve a few crystals in chloroform, add concentrated sul- 
phuric acid, and shake the mixture : the chloroform assumes a blood- 
red to a purplish-red color, while the sulphuric acid at the same time 
shows marked fluorescence. 

To isolate the fatty acids, the solution of soaps obtained above is 
acidified with dilute sulphuric acid, when the fatty acids which have 
separated out may be filtered off and identified individually by their 
boiling-points and the analysis of their barium salts. 

The final filtrate, when neutralized with ammonium hydrate, con- 
tains glycerin. 

The Biliary Acids. — The biliary acids found in the feces are : 
glycocholic acid (C 26 H 43 N0 6 ), taurocholic acid (C 26 H 45 NS0 7 ), and 
cholalic acid (C^H^O.). 

The two former occur normally in the bile, and can be decomposed 
into cholalic acid and glycocoll, and cholalic acid and taurin, respec- 
tively ; as this process of decomposition takes place ordinarily in the 
intestines, the third acid — i. e., cholalic acid — is always found in the 
feces. 

In order to demonstrate the biliary acids, the fatty acids, phenols, 
indol, and skatol are first removed by distillation with phosphoric 
acid. The residue is taken up with water and boiled, and the filtered 
liquid precipitated with lead acetate and a little ammonium hydrate. 
The biliary salts of lead are contained in the precipitate, from which 
they can be removed by washing with water and finally boiling the 
precipitate with alcohol. The washings are filtered and the lead salts 
transformed into sodium salts by treating the filtrate with sodium 
carbonate. After further filtration the filtrate is evaporated to dry- 
ness and the residue extracted with hot alcohol. Upon evapora- 
tion the salts of the acids sometimes crystallize out as such, Avhile 
more often a dirty amorphous precipitate is obtained, which may be 
rendered crystalline by treating with ether. The amorphous residue, 
however, can be employed for making the necessary tests. 

Pettenkofer's Test. — A small amount of the substance is dissolved 
in water, and treated with two-thirds its volume of concentrated 
sulphuric acid, care being taken that the temperature does not exceed 
60° or 70° C. While stirring, a 10 per cent, solution of cane-sugar 
is added drop by drop. If biliary acids are present, the solution 
assumes a beautiful red color, which on standing turns a bluish violet. 
This test depends upon the action of furfurol, derived from the sul- 
phuric acid and cane-sugar, upon the biliary acids. 

Pigments. — The principal pigment of normal feces is termed 



284 THE FECES. 

stercoLilin, and was first isolated from this source by Vanlair and 
Masius. 1 Owing to its great similarity to hydrobilirubin, it has even 
been regarded as identical with it, but Garrod and Hopkins 2 have 
now conclusively shown that whereas the urobilin of the urine and 
the stercobilin of the feces are identical in composition, as also in 
properties, they differ conspicuously from hydrobilirubin, and espe- 
cially in the much smaller percentage of nitrogen which they con- 
tain, viz., 4.11, as compared with 9.22 per cent. It is derived from 
bilirubin, and formed in the upper regions of the large intestine 
more especially, as the result of bacterial activity. 3 This explains 
the observations that as a rule the meconium and the solid excreta 
of the first day or two of life contain no urobilin, and that the 
pigment also disappears, when for any reason the bile is prevented 
from entering the intestinal canal. 

To isolate the pigment from the feces, the material is first extracted 
with alcohol. The alcoholic extract is evaporated to dryness ; the 
residue is extracted with water, the aqueous solution acidified with 
sulphuric acid and saturated with ammonium sulphate, when on 
shaking with chloroform or a mixture of chloroform and ether the 
pigment is taken up by the organic solvent. 

The free pigment is a brown amorphous substance of a character- 
istic odor, and melts at a temperature below 100° C. On cooling, 
it forms a brittle, shellac-like material, which is said to be quite char- 
acteristic. It is soluble in ether, chloroform, water, and amyl 
alcohol. On treating its solutions with zinc chloride and ammonia 
a beautiful green fluorescence is obtained. Such solutions then show 
three bands of absorption, of which the one between C and F is the 
most characteristic (see also Urinary urobilin). 

Haematoporphyrin, to judge from recent investigations by Stokvis 4 
and Garrod, 5 is likewise a normal component of the feces, but oc- 
curs only in traces. Garrod states that with Saillet's 6 method, the 
basis of which is extraction with acetic ether, after the addition of 
acetic acid, he invariably found traces, comparable with those which 
normally are present in the urine. He also states that he found 
considerably larger amounts of the pigment in the meconium, both 
in that expelled during the first day or two of life, and in that re- 
moved from the intestines of stillborn infants. 

The presence of these normal traces has been referred by some to 
the ingested blood-coloring matter of red meat and vegetable chloro- 

1 Vanlair and Masius, Centralbl. f. d. med. Wiss., 1871, vol. ix. p. 369. 

2 F. G. Hopkins and A. E. Garrod, " On Urobilin," Jour, of Physiol., 1898, vol. xxii. 
p. 451. 

3 A. Schmidt, Verhandl. d. XIII. Congresses f. inn. Med., 1895, p. 320. Vaughan 
Harley, Brit. Med. Jour., 1896, vol. ii. p. 898. Macfadyen, Nencki, and Sieber, Arch, 
f. exper. Path. u. Pharmakol., 1891, vol. xxviii. p. 311. 

4 Stokvis, Nederl. Natuur-en Geneeskundig Congres, 1899, p. 378. 

5 Garrod, "The Urinary Pigments in their Pathological Aspects," Lancet, Nov. 10, 
1900. 

6 Saillet, Rev. deMed,, 1896, vol. xvi. p. 542. 



PATHOLOGY OF THE FECES. 285 

phyll. Garrod, however, finds that the hsernatoporphyrin does not 
disappear when these articles of diet are withdrawn, and while 
admitting that the ingested haemoglobin and chlorophyll may possi- 
bly be converted, in part at least, into hseniatoporphyrin, he concludes 
that the greater portion is derived from human sources. On the 
whole, the evidence seems now in favor of the view that the hsemato- 
porphyrin which is found both in the urine and in the feces originates 
within the liver, and is eliminated into the intestinal canal in the 
bile (see also Hsematoporphyrinuria). 

Xanthin Bases. 1 — The amount of xanthin bases which is normally 
eliminated in the feces is about 0.11 gramme. They are largely 
represented by guanin, and adenin, while xanthin and hypoxanthin 
are the common forms which are met with in the urine. 

PATHOLOGY OF THE FECES. 

General Characteristics. 

Number of Stools. — As has been pointed out (page 207), one or 
two stools in the twenty-four hours may be considered as normal. 
Individual peculiarities, however, must be taken into consideration. 

As the consistence of the stools is altered in diarrhoea, this condi- 
tion may be defined as one in which too frequent and liquid passages 
occur, while the reverse holds good for constipation, the consistence 
of the stools in this condition being usually also altered. The term 
obstruction, on the other hand, denotes a state of affairs in which 
no stools are voided. In a general way it may be said that what- 
ever gives rise to increased peristalsis likewise produces diar- 
rhoea, and that whatever diminishes peristalsis gives rise to con- 
stipation. In the former condition the number of stools may vary 
from one to thirty, forty, or even fifty in the twenty-four hours, 
as in Asiatic cholera. The consistence of the stool when only one 
is passed in the twenty-four hours will, of course, decide the ques- 
tion whether the case should be regarded as one of diarrhoea or not. 
One stool passed in the twenty-four hours may under certain condi- 
tions be regarded as a symptom of constipation, but more commonly 
this term is applied to a condition in which a stool occurs only every 
two, three, four, or more days. 

Consistence and Form. — The consistence of the stools may 
undergo variations, w r hich run a course parallel to their number. 
They may be thin, mushy, and even watery. 

In constipation, on the other hand, owing to an increased absorp- 
tion of water, the feces may be passed as very hard and perfectly 
dry, roundish, scybalous masses, the rotundity of which is undoubt- 
edly referable to their long sojourn in the haustra of the colon. The 

1 M. Kriiger u. A. Schittenhelm, Zeit. f. phys. Chem., 1902, vol. xxxv. p. 753. 



286 THE FECES. 

individual scybala usually vary in size from that of a hazelnut to that 
of a walnut, and are frequently provided with one or two indenta- 
tions which represent impressions of the taenia of the colon. Still 
smaller masses, closely resembling the dejecta of sheep, may also be 
seen. Their presence was formerly regarded as characteristic of 
stricture of the colon, but they are likewise found in ordinary cases 
of chronic constipation. Fecal ribbons and columns of the diame- 
ter of a pencil are found in cases of enterospasm of neurotic origin, 
as well as in stricture of the colon. 

Amount .^-The absolute amount of feces voided in the twenty- 
four hours bears an inverse relation to the number of stools and their 
consistence, providing, of course, that no abnormally large ingestion 
of food has occurred. In that case an abnormally large stool of 
moderate firmness may be passed. Two exceptions must, however, 
be noted to this rule — i. e., the passage of large quantities of firm 
feces, following an attack of constipation of long duration or an 
attack of obstruction. Lynch 1 reports a remarkable instance in 
which, following a prolonged attack of constipation, an enema 
caused the evacuation of 20 kgrms. of fecal matter. Especially 
large amounts of feces are observed in cases of biliary obstruc- 
tion, where 1100 grammes may be exceeded. In cases of fer- 
mentative dyspepsia the amount may also be large, varying be- 
tween 400 and 900 grammes, while the patients are on a diet on 
which normal individuals would pass from 200 to 270 grammes 
in the twenty-four hours. Still larger amounts are noted in cases 
of enteritis. Schmidt mentions a case in which 2780 grammes were 
eliminated (these figures have reference to a three days' experiment 
with a test diet ; see page 294). 

Odor. — As the normal offensive odor of the feces is largely due to 
products of intestinal putrefaction, an increase in this respect will 
naturally be referable to conditions in which the putrefactive proc- 
esses are increased. A most disagreeable odor is thus met with in 
the so-called acholic stools. The odor of fatty acids is observed 
in the lighter grades of infantile diarrhoea, while a markedly putrid 
odor is associated with its severer forms. A very characteristic, 
sperm-like odor is further noted in the stools of cholera, owing to the 
presence of considerable quantities of cadaverin. A truly rotten 
stench is present in the gangrenous form of dysentery, and in car- 
cinomatous and syphilitic ulceration of the rectum. An ammoni- 
acal odor is due to an admixture of urine undergoing ammoniacal 
decomposition. 

Reaction. — The reaction of the stools is variable under patho- 
logical conditions, and of but little clinical interest. In typhoid 
fever an alkaline reaction is so constantly met with that this symp- 
tom might possibly be of value in doubtful cases. It may, however, 

X E. Lynch, " Coprologia," Thesis, Buenos- Ayres, 1896, p. 38. 



PATHOLOGY OF THE FECES. 287 

also be neutral, amphoteric, or even acid. In acute infantile diar- 
rhoea an acid reaction is the rule, but exceptions also are not infre- 
quent. Normal stools of sucklings are acid, the degree of acidity, 
according to Langstein, 1 corresponding to about 2.1 to 3.7 per cent, 
of normal NaOH for 100 grammes of the moist feces. 

Color. — The color of the feces in disease may vary a great deal. 
When unaltered bile is present, the stools may assume a golden- 
yellow, a greenish-yellow, or even a green color. In cases of biliary 
obstruction or suppression, on the other hand, they become pasty 
and have a grayish or even a white color. This, however, is not so 
much due to the absence of coloring-matter derived from the bile, as 
to an insufficient absorption of fats, as was shown by Strunipell, who 
succeeded in obtaining stools of a light-brown color after feeding 
patients affected with catarrhal jaundice upon a diet containing 
minimal amounts of fat. Such acholic or colorless stools, as it would 
be better to say, are not only found associated with biliary obstruc- 
tion, however, but may also occur when the ducts are patent. They 
have thus been observed in various cases of leukaemia, carcinoma of 
the stomach or intestine, in simple infantile enteritis, chronic nephri- 
tis, chlorosis, scarlatina, tubercular enteritis, and especially frequently 
in debilitated consumptives and in cases of chronic tubercular peri- 
tonitis in children. In some of these conditions, as in tuberculosis 
of the intestines and of the peritoneum, the lack of color is probably 
due to a diminished absorption of fats. In others, however, this 
explanation does not hold good, as abnormally large amounts of fat 
are not necessarily present. In such cases the lack of color is prob- 
ably referable to the formation of colorless decomposition-products 
of bilirubin, such as the leuko-urobilin of Nencki, but nothing 
definite is known of the conditions which favor the formation of 
these products. In this connection it may be interesting to note 
that in those cases in which the biliary ducts are patent the color of 
the stools may Vary not only from day to day, but even within the 
twenty-four hours. 1 A neurasthenic patient occurring in my prac- 
tice thus passed an acholic stool almost every morning and usually 
colored feces in the afternoon, for a period of several weeks. 

Generally speaking, the color of the stools becomes lighter the 
larger the number of movements, and vice versa. In Asiatic cholera 
and dysentery they may thus be colorless, while in severe constipa- 
tion the scybalous masses are almost black. 

If blood is present, the stools may present a scarlet-red, a dirty 
brownish-red, a coffee, or even a perfectly black color. Adherent 
blood, usually bright red in color and found on scybalous masses, is 
probably always derived from the rectum or anus, while a change in 

1 L. Langstein, Jahrbuch. f. Kinderheilk., vol. vi. Heft 3. 

2 Pel, Centralbl. f. klin. Med., 1887, vol. viii. p. 297. Le Nobel. Arch. f. klin. Med., 
1888, vol. xliii. p. 285. Vogel-Biedert, Lehrbuch der Kinderkrankheiten, 9th ed., 1887, 
p. 115, Enke, Stuttgart. Berggriin u. Katz, Wien. klin. Woch., 1891, vol. iv. p. 858. 



288 THE FECES. 

color, indicating an earlier date of the bleeding, usually points to 
the colon. 

An intimate admixture of blood with the stool, the color being at 
the same time altered, so as to vary from a brownish red to black 
(owing to the presence of ferrous sulphide), is indicative of hemor- 
rhage into the stomach or the small intestine. The darker the color 
of the blood the more remote from the anus will be, as a rule, the 
seat of the hemorrhage. Black or coffee-colored stools are thus 
observed in cases of ulcer of the stomach or of the duodenum, in 
melsena neonatorum, and similar conditions. 

When profuse intestinal hemorrhages take place, however, as in 
some cases of typhoid fever and melaena, and particularly when 
diarrhoea exists at the same time, the blood which appears in the 
stools may be changed very little or not at all. 

While, as a rule, simple inspection or a microscopical examination 
of the feces will determine whether or not blood is present, it may 
at times be necessary to resort to more delicate tests, as the hemor- 
rhage may have been so slight as to escape detection with the naked 
eye, or so far removed from the anus that even blood-shadows can- 
not be found with the microscope. Hemorrhages of such trivial 
extent have been reported by Hasslin as occurring quite frequently 
in cases of chlorosis. This statement, however, I have not been 
able to cou firm. If an investigation in this direction is to be made, 
the method of Muller and Weber (see page 261), or that of Kor- 
czynski and Jaworski, should be employed. 

Korczynski and Jaworski's Test. — A small amount of the fecal 
material is treated with a pinch of potassium chlorate and a drop of 
concentrated hydrochloric acid. The mixture is carefully heated 
until it has become decolorized, more hydrochloric acid being added 
if necessary. The chlorine is then driven off, when one or two drops 
of a dilute solution of potassium ferrocyanide are added. In the 
presence of blood-coloring matter a distinct blue color is obtained, 
owing to the formation of Prussian blue. 

For most purposes, however, the hsemin test will suffice, espe- 
cially with the modification suggested by Strzysowski. The darkest 
particles of feces are boiled with a drop of a sodium iodide solution 
(1 : 500) and concentrated acetic acid. The resulting hsemin crys- 
tals are of a darker color than the common form (the hydrochlorate). 

An admixture of pus in notable amounts also gives rise to a 
characteristic color, as is seen in cases of dysentery, syphilitic and 
carcinomatous ulceration of the colon and rectum, following the per- 
foration of a parametritic or periproctitic abscess into the rectum, etc. 

Carter and MacMunn l have recently pointed out that at times a 
chromogen may be present in the feces, which on exposure to the 
air is transformed into a red pigment, simulating blood-coloring 
matter. They report three cases in which this was observed. Mac- 

1 Carter and MacMunn, Brit. Med. Jour., 1899. 



PATHOLOGY OF THE FECES. 289 

Munn expresses the opinion that the substance in question is closely 
related to stercobilin. The stools showed streaks of red upon the 
surface, and after further exposure and repeated agitation turned a 
pronounced blood red throughout. 

Green stools are observed especially in infants, and may be refer- 
able to two different causes, being dependent on the one hand upon 
the presence of a bacillus, described by Le Sage, which produces a 
green coloring-matter, while on the other it may be referable to 
biliverdin. When green stools occur frequently, this condition is 
associated with the clinical symptoms of a severe cholera infantum. 
Such stools have also been noted in dysentery referable to an infec- 
tion with the Bacillus pyocyaneus. 

Quite characteristic also are the ipecacuanha stools, which closely 
resemble the so-called acholic stools. The green color produced by 
calomel, the yellow by santonin, rheum, and senna, the black by 
iron, manganese, and bismuth, have already been mentioned (see 
page 208). 

Macroscopical Constituents. 

Alimentary Constituents. — After having thus considered the 
number of stools, their consistence, reaction, odor, and color, it is 
necessary to look for gross admixtures, and especially for the presence 
of undigested food-material, such as pieces of meat, flakes of casein 
— this especially in the stools of children — and fragments of amyla- 
ceous food. The occurrence of such a condition, constituting what 
was formerly known as lientery, is always indicative of disturbed 
intestinal or gastric digestion, or both. It is, hence, observed in 
cases of chronic intestinal catarrh, febrile dyspepsia, following the 
use of cathartics, etc. 

Occasionally also unaltered food in large amounts is found in the 
feces, owing to a direct communication between the stomach and the 
colon, as in cases of perforating ulcer or carcinoma of the stomach. 

When fat is present in abnormally large amounts it can usually be 
recognized with the naked eye. To this condition the term steatorrhea 
has been applied (see page 287). In typical cases the fat is seen in 
the form of whitish or grayish masses, varying in size from that of a 
pea to that of a walnut, which are more or less intimately mixed with 
the fecal material, and may at first sight be mistaken for flakes of 
casein. From these, it may be distinguished, however, by its chem- 
ical reactions and its peculiarly glistening appearance. In other 
cases stools may be seen in which the fecal column is covered, to a 
greater or less extent, with a grayish, dense, asbestos-like substance, 
while the core itself presents the usual color. Nothnagel states that 
this appearance is referable to congealment of the fat, when it is 
exposed to a lower temperature than that of the body. I have re- 
peatedly observed this appearance, however, in stools which had just 
been voided and were still warm. The passage of liquid oil in the 
19 



290 THE FECES. 

absence of fecal material has also been recorded, but it seems doubt- 
ful that the oil in such cases entered the body by the mouth. Fol- 
lowing the use of oil enemata such stools are, of course, seen. 

The elimination of abnormally large quantities of fat may be due 
to the ingestion of correspondingly large amounts. More frequently, 
however, it is referable to distinctly pathological conditions. A 
steatorrhea will thus naturally occur when an insufficient supply 
of bile is poured into the small intestine, and hence is observed 
constantly in cases of biliary obstruction. In these cases, however, 
the microscope is usually necessary to demonstrate the presence of 
the abnormally large quantities of fat. True steatorrhea, on the 
other hand, viz., the presence of fat recognizable with the naked 
eye, is more commonly met with in diseases affecting the resorptive 
power of the small intestine, such a*s extensive atrophy or amyloid 
degeneration of the intestinal mucosa, tubercular ulceration, etc., or 
in diseases involving the integrity of the lymphatic glands and vessels 
of the mesentery, as in chronic tubercular peritonitis, caseous degen- 
eration of the mesenteric glands, etc. In simple catarrhal condi- 
tions, however, steatorrhoea may also occur, and not only in infants, 
but, according to my experience, also in adults. The question 
whether or not steatorrhoea is constantly observed in cases of pan- 
creatic disease, as some observers have claimed, may now be answered 
in the negative, although it must be admitted that the two conditions 
are very frequently associated. Le Nobel, who has recently inves- 
tigated this subject, arrived at the conclusion that the steatorrhoea in 
itself is of little practical importance, but that its association with 
the absence of products of putrefaction from the stools, the absence 
of the salts of the fatty acids, and the presence of maltose in the 
urine, may possibly be regarded as indicating the existence of pan- 
creatic disease. 

Mucus and Mucous Cylinders. — So long as mucus occurs in 
small particles only, adherent to otherwise normal feces, it is of no 
pathological significance. Larger amounts are almost always indic- 
ative of a catarrhal condition of the colon or rectum, no matter 
whether the stool is otherwise normal or whether diarrhoea exists at 
the time. Peculiar formations are occasionally seen, viz., so-called 
mucous cylinders, which are passed in large or small fragments in a 
condition which has been described by Nothnagel as enteritis mem- 
branosa or colica mucosa} Such masses, which at times measure a 
foot or more in length, are ribbon- or net-shaped, and are frequently 
passed in the absence of fecal matter, with severe tenesmus. They 
closely resemble Curschmann ? s spirals, but lack the central thread 
and the Charcot-Leyden crystals. They are probably indicative of 

1 Nothnagel, "Colica mucosa," Beitrage z. Physiol, u. Path. d. Darmes, 1884. 
Fleiner, Berlin, klin. Woch., 1893, Nos. 3 and 4. ~ Einhorn, Arch. f. Verdauungs- 
krank., vol. iv. p. 456, 



PATHOLOGY OF THE FECES. 291 

chronic constipation associated with catarrh of the colon. Not to 
be confounded with this condition is the passage of masses of mucus, 
which do not present the cylindrical form, but which also may be 
passed with a great deal of tenesmus and in the absence of fecal 
matter ; this is very commonly seen in cases of nephroptosis asso- 
ciated with gastroptosis and enteroptosis. These formations are in 
all probability also referable to a catarrhal condition of the colon. 
In cholera Asiatica particles of mucus are seen which resemble 
grains of rice ; their presence was formerly regarded as characteristic 
of the disease ; but they are now known to occur in ordinary catar- 
rhal conditions also. 

Biliary and Intestinal Concretions. — Most important from a 
diagnostic standpoint is the examination of the feces for the presence 
of biliary concretions, which should never be neglected in cases of 
colicky abdominal pain of doubtful origin, whether associated with 
jaundice or not. 

When searching for gall-stones the feces should be stirred with 
water and passed through a fine sieve. Biliary concretions may then 
be found as small, crumbling masses or as hard stones presenting an 
irregular contour or the smooth, characteristic facets. In size they 
may vary from that of a millet-seed to that of a pigeon's egg ; large 
stones are rarely passed by the bowel unless perforation has occurred 
into the intestines and usually into the colon. 

Some calculi consist almost entirely of cholesterin, while others 
are composed essentially of inspissated bile, and still others of 
calcareous salts. The former are the most common, and are 
readily recognized by their softness and color, which may be white, 
grayish, bluish, or greenish. Their specific gravity is lower than 
that of water. Very frequently they contain a nucleus, composed 
of earthy sulphates or phosphates. An analysis which I made of 
a stone of this kind, weighing 10.548 grammes, gave the following 
results : 

Cholesterin 72.590 per cent. 

Mineral salts 0.247 " 

Fats 5.090 " 

Biliary pigments ] 3.930 " 

Organic matter 7.270 " 

Calculi which consist largely of biliary pigments are brown in 
color. They are hard, and heavier than water. Frequently they 
contain traces of copper and zinc (Fig. 54). 

Calculi composed of calcareous salts generally present an irregular, 
roughened contour. 

Within recent years Welch has drawn attention to the not infre- 
quent presence of pure colonies of the Bacillus coli communis in gall- 
stones, apparently forming their nucleus. Typhoid bacilli also have 
since been observed in their interior, and it appears likely that the 



292 THE FECES. 

formation of gall-stones is primarily referable to an invasion of the 
gall-bladder by such micro-organisms. 

Analysis of Gall-stones. — The stone is finely powdered and dried 
at a temperature of 100° C. It is then extracted with boiling water 
and the washings concentrated upon a water-bath to about 100 c.c. 
One portion of this amount is evaporated to dryness, and the soluble 
residue, as well as the mineral ash, determined after desiccation at a 
temperature of 105° C. The other portion is likewise evaporated 

Fig. 54. 




Gall-stones, 
a, cholesterin ; 6, pigment-stones. 

to dryness and extracted with alcohol containing a small amount of 
ether, sodium glycocholate being thus obtained. After treatment 
with hot w r ater, as described, the substance is successively extracted 
with alcohol and ether. In the alcoholic extract fats and a small 
amount of cholesterin will be found. The greater portion of this 
is in the ethereal extract. The residue, which is insoluble in hot 
water, alcohol, and ether, is treated with a moderately strong solu- 
tion of hydrochloric acid, the earthy phosphates and oxides being 
thus obtained united to pigments. The bilirubin is removed by 
extracting with boiling chloroform. The pigments which are not 
dissolved in this manner are biliprasin, bilihumin, etc. 

Intestinal concretions (enteroliths) are rare and usually come from 
the appendix. At times they contain some foreign body, such as a 
grape-seed, as a nucleus, upon which calcium and magnesium salts 
have become deposited. 

Fecal calculi or coproliths are likewise only rarely seen. They 
represent inspissated fecal material which has become impregnated 
with lime and magnesium salts. More commonly they are found at 
the post-mortem table in the caecum, in the haustra of the colon, 
and in the rectum. 

Intestinal sand is also rare. In the German literature I have 
found reports of only three cases, while in the French literature 
about sixteen have been recorded. Of its origin nothing is known. 
The condition is commonly associated with enteritis membranacea. 
The material presents a brownish color, but may be light green. 
In one case reported by Deetz * it was possible to demonstrate the 
presence of calcium phosphate with traces of calcium oxalate. 

1 E. Deetz, Deutsch. Arch. f. klin. Med., 1901, vol. lxx, p. 365. See also Dieulafoy, 
"La Hthiase intestinale et la gravelle de l'intestin," Presse med., March 10, 1897 
(extract in Centralbl. f. klin. Med., 1897, p. 904). 



PATHOLOGY OF THE FECES. 293 

Microscopical Examination. 

Technique. — In hospital work the stool should be passed into a 
well-warmed bed-pan and examined at once. This is particularly 
important in the search for amoebae. In private practice patients 
should be instructed to send their stools to the physician as soon as 
possible, when suspicious-looking particles should be placed upon 
the warm stage or examined upon a well-warmed slide. A very 
convenient form of warm stage, which may be obtained from instru- 
ment-makers at low cost, is composed of brass and made to be held 
in position on the stage of the microscope by spring clips. It is 
about 8 cm. long and 3 cm. broad, and has cemented to a recessed 
bottom an ordinary glass slip; an opening measuring 1.35 cm. in 
diameter is in the centre of the stage. To one of the long sides of 
the brass stage is fitted a projecting stem, about 10 cm. long, to 
which the heat of a spirit-lamp is applied. 

For ordinary purposes it is well to place the stool, if watery, 
in a conical glass, and to cover it with a layer of ether, so as to 
diminish the disagreeable odor. If mushy or firm, it should be 
spread upon a plate and covered with a layer of turpentine, or a 
5 per cent, solution of carbolic acid or thymol. 

Remnants of Food. — It has been pointed out that various micro- 
scopical remnants of food are observed in normal feces. In patho- 
logical conditions it is necessary to determine whether or not such 
remnants are present in abnormal amount, presupposing, of course, 
that excessive quantities of food have not been ingested. It is often 
possible to draw definite conclusions as to the state of intestinal 
digestion from the excess of one form of non-digested material over 
another. The presence of large quantities of undigested starch 
indicates a catarrhal condition of the small intestine, and it may, 
indeed, be said that the occurrence of more than traces of this mate- 
rial should always be regarded with suspicion. An increase in the 
number of muscle-fibres will, as a rule, likewise be observed under 
such conditions. 1 

Schmidt and Strassburger 2 have described a special form of intes- 
tinal fermentative dyspepsia, in which there is an isolated amylo- 
lytic insufficiency, which may be of functional or of organic origin 
(see Schmidt's fermentation test below). 

In this connection it is noteworthy that in man extensive disease 
of the pancreas may exist without seriously disturbing amylolytic 
digestion. 

Schmidt's Fermentation Test. — To obtain a more exact insight into 
the degree of amylolytic insufficiency of the intestinal tract than 

1 Schmidt u. Strassburger, Deutsch. Arch. f. klin. Med., vol. lxix. p, 570. 

2 A. Schmidt, "Die Klinische Bedeutung der Ausscheidung von Fleischresten mit 
dem Stuhlgang," Deutsch. med. Woch., 1899, p. 811. 



294 



THE FECES. 



is possible from a microscopical study of the feces, Schmidt has pro- 
posed a special method which is based upon the continued diges- 
tion of the carbohydrates in the feces. The examination is made 
after the patient has been placed on the following test diet (Schmidt 
and Strassburger's test diet No. II.): milk, 1.5 liters; 3 J eggs; 
strained oatmeal gruel (from 80 grammes of oatmeal) ; 100 grammes 
of Zwieback; 20 grammes of butter; 20 grammes of sugar; 125 
grammes of steak (raw weight) ; and 190 grammes of potato (raw 
weight). The distribution of these various articles of food can 
be arranged as one chooses, or as follows : at 7.30 a.m., -| litre 
of milk and 2 Zwiebacks (each 33 grammes) ; at 10.30 A.M., 



-f litre of bouillon with J 



egg; 

i 



at 12 M. -I litre of milk with 1 



Fig. 55. 




egg ; between 1 and 2 p.m. J litre of oatmeal gruel (prepared from 
40 grammes of oatmeal, 166 grammes of milk, 10 grammes of sugar, 
and J egg) ; 100 grammes of well-done Hamburg steak (125 
grammes of raw beef (raw weight) and 12 grammes of butter ; 250 
grammes of mashed potato (from 190 grammes 
of potato, 60 grammes of milk, and 8 grammes 
of butter) ; at 4.30 p.m. -| litre of milk, 1 egg, 
1 Zwieback ; at 7.30 p.m. J litre of oatmeal 
gruel as at dinner-time. Before commencing 
with the test diet, however, it is necessary to 
demarcate the fecal material by giving a wafer 
or capsule containing 0.3 gramme of powdered 
carmine. The examination proper is made as 
soon as the feces are no longer colored red, viz., 
after from two to three days of the test diet. 
The necessary apparatus is pictured in the accom- 
panying figure (Fig. 55), which represents one- 
third of the actual size. For each experiment 
5 grammes of fresh fecal material are used (the 
feces being of medium consistence ; otherwise a 
little more or less is taken, corresponding to 
about 1 gramme of dry residue). The material 
is well stirred with water in the bottle a, which 
is filled entirely and closed with the rubber stop- 
per, care being taken to exclude bubbles of air. 
Tube b is filled with water from the tap and 
also closed without admission of air. Tube c 
should contain no water ; it has a pin-hole aper- 
ture at the top. The communicating tube d is 
adjusted as shown in the figure. The apparatus is 
then placed in the incubator at 37° C. for twenty- 
four hours, not longer. During this time the 
carbohydrate fermentation will have been completed (Schmidt's 
Fruhgahrung). During the evolution of gas water will be dis- 



Sehmidt's fermentation 
tubes. 



PATHOLOGY OF THE FECES. 295 

placed from b into o; the resulting column is measured and repre- 
sents the degree of fermentation. The result is regarded as positive 
if more than a quarter tubeful of gas is obtained. With the test 
diet in question this would mean a condition approximating the 
normal. In such an event the patient is placed for two days fur- 
ther on test diet No. I., which differs from No. II. only in the 
absence of the meat and potato. If then there is still a positive 
result, the diagnosis of " fermentative dyspepsia " is justifiable. In 
order to eliminate errors arising from possible formation of gas as 
the result of albuminous putrefaction the fermenting fecal material 
should be tested from time to time in a control specimen. If the 
formation of gas is due to carbohydrate fermentation, there will be an 
increasing degree of acidity (tested with litmus-paper) ; this increase, 
however, is not always marked ; at any rate, there must be no 
increasing alkalinity. 

The so-called acholic stools are usually very rich in fat, and particu- 
larly so in cases of biliary obstruction associated with jaundice. At 
other times the lack of color, as has been mentioned above, is not 
referable to the secretion of an insufficient amount of bile, but to 
the presence of colorless decomposition-products of bilirubin, such 
as the leuko-urobilin of Nencki. In these cases abnormally large 
quantities of fat are not always, present. The conclusion that a 
stool contains excessive amounts of fat because it is apparently 
acholic is hence not justifiable unless a microscopical examination 
has been made. 

Leiner's Test for Casein. — Casein is most conveniently demon- 
strated with Leiner's method. To this end, a small amount of 
fecal matter is spread on a slide and dried in the air. It is then 
fixed by heat — passing the specimen through the flame of a Bunsen 
burner three or four times is sufficient — and stained with a mixture 
of equal parts of a 0.75 per cent, solution of ' acid fuchsin and 
methyl-green in 50 per cent, alcohol, the mixture being diluted ten 
times with water. After fifteen minutes the preparations are placed 
in distilled water and allowed to remain for one hour or longer. 
Casein and paracasein are thus stained a pale blue or violet, while 
similar bodies are practically all colored a light green, or more rarely 
a yellowish green. 

Epithelium. — Epithelial cells when present in large numbers 
always indicate an inflammatory condition of some portion of the 
intestinal tract. 

Cylindrical epithelial cells are found in abundance in all inflam- 
matory conditions affecting the intestinal mucosa. They are almost 
exclusively seen imbedded in mucus, and it is interesting to note that 
the cloudy appearance of the mucus is referable to the presence of 
these elements, and not to leucocytes, as is the case in the sputum. 
When bile-stained specimens are met with, the conclusion is justifi- 



296 THE FECES. 

able that the small intestine is involved. Degenerative forms are 
mostly seen ; well-preserved cylindrical or goblet-cells may, however, 
also be found, and are, according to my experience, much more com- 
mon than is generally supposed. 1 

Epithelioid cells may be found in carcinoma of the rectum. 

Red Blood-corpuscles. — Unaltered red blood-corpuscles, accord- 
ing to Nothnagel, are but rarely observed in the feces, no matter 
how intensely red they may be colored, providing that an ulcerative 
process affecting the colon or the rectum can be excluded ; in that 
case, as in the severer forms of dysentery, large numbers may be 
observed. If the hemorrhage has occurred higher up in the intes- 
tine, large and small masses of a brownish-red color are seen, which 
consist of hsematoidin. They are mostly amorphous, but in some 
specimens the characteristic rhombic crystals may be observed. In 
general, it may be said that the higher the seat of the hemorrhage 
the darker will be the color of the pigment, and the less the chance 
of finding well-defined red corpuscles. In such cases recourse musi 
be had to the hsemin test (page 39), to the iron test of Korczynski 
and Jaworski (page 288), or to Donogany's test (page 262). 

Mucus. — Small hyaline particles of mucus, visible only with the 
microscope, are not infrequently met with under pathological condi- 
tions, and are of distinct diagnostic significance. When bile-stained, 
their presence is always indicative of disease of the small intestine 
proper, while colorless particles point to a catarrhal condition of the 
upper portion of the large intestine or the lower portion of the small 
intestine. Beginners should be careful not to mistake apparently 
hyaline particles of vegetable residue for mucus. Mucus never yields 
a blue color when treated with iodine, or iodine and sulphuric acid, 
and examination with a higher power will show the entire absence 
of any definite structure. Both forms, viz., colorless and colored 
particles, are found intimately mixed with the feces, and may be very 
abundant. In addition to these forms Nothnagel has described the 
occasional occurrence of large numbers of roundish or irregular, 
very pale hyaline or opaque formations, which are devoid of all 
structure. Some specimens are homogeneous, while others present a 
distinct rimous appearance. They have thus far been found only in 
liquid stools, and are apparently of no diagnostic significance. To 
judge from their optic behavior, they probably consist of mucus. 2 

Leucocytes. — The presence of a large number of leucocytes usu- 
ally indicates a severe catarrhal, if not an ulcerative, condition of the 
intestines, the number of leucocytes or pus -corpuscles standing in a 
direct relation to the intensity of the inflammatory process. Pure 
pus in large amounts is observed especially in dysentery, and in 

1 Nothnagel, loc. cit., page 226. 

2 A. Schmidt, " Ueber Schleim im Stuhlgang," Zeit. f. klin. Med., vol. xxxii. p. 260. 



PATHOLOGY OF THE FECES. 297 

cases in which accumulations of pus have perforated into the gut 
from adjacent organs or cavities. 

Crystals. — The crystals which may occur in the feces have already 
been briefly considered (page 274). Of these, the so-called Charcot- 
Leyden crystals deserve more detailed consideration. While occur- 
ring at times in normal stools, as also in those of typhoid fever, 
dysentery, and phthisis, such observations are rare. They appear to 
be quite constantly present, on the other hand, in cases of ankylo- 
stomiasis and anguilluliasis. They are also frequently associated with 
Ascaris lumbricoides, Oxyuris vermicularis, Taenia solium and sagi- 
nata. In cases of Trichocephalus they are but rarely seen, while they 
are always absent in the case of Taenia nana. These observations, 
made by Leichtenstern, are very important, and, according to the same 
observer, the occurrence of Charcot-Leyden crystals should always 
excite suspicion as to the existence of helminthiasis and lead to a 
careful examination of the feces for parasites or their ova. Their 
persistence in the feces after the evacuation of what would appear 
to be a complete taenia should be regarded as indicating the non- 
removal of the head. In amoebic colitis these crystals have also 
been observed by Lewis, Laileur, Amberg, myself, and others. 1 



Animal Parasites. 

I. — Protozoa : 

1. Rhizopoda, 

Monera, 

Amoebina : Amoeba coli. 

2. Sporozoa, S. gregarina, 

Coccidia, 

Plasmodium nialariae. 
•3. Infusoria, 

a. Ciliata, 

Holotricha : Balantidium coli. 

b. Flagellata. 

Monadina, 

Cercomonadina : Cercomonas. 
Isomastigoda. 

Tetramitina : Trichomonas. 
Polymastigina : Megastoma. 
II. — Vermes : 

Platodes, 

Cestodes, 

Taenia saginata. 

Taenia solium. 

Taenia nana. 

Taenia diminuta. 

Taenia cucumerina. 

Bothriocephalus latus. 

Krabbea grandis. 

1 Leichtenstern, Deutsch. med. Woch., 1885, vol. xi. Nos. 29 and 30; Ibid., 1886, 
vol. xii. Nos. 11-14 ; Ibid., 1887, pp. 565, 594, 620, 645, 669, 691, aud 712. 



298 THE FECES. 

II. — Vermes {continued) : 
Trematodes, 

Distoma hepaticum. 
Distoma lanceolatum. 
Distoma Buskii. 
Distoma sibiricum. 
Distoma spatulatum. 
Distoma conjunctum. 
Distoma heterophyes. 
Amphistoma hominis. 
Distoma haematobium. 
Distoma pulmonale. 
Annelides, 

Nematodes, 
Asearides, 

Ascaris lumbricoides. 
Ascaris mystax. 
Ascaris maritima. 
Oxyuris vermicularis. 
Strongyloides, 

Anchylostomum duodenale. 
Trichotrachelides, 

Tricliocephalus hominis. 
Trichina spiralis. 
Ehabdonema strongyloides, 
Anguillula intestinalis. 
III. — Insecta : 

Piophila casei. 

Drosophila melanogastra. 

Homialomyia. 

Hyodrothoea meteorica. 

Cystoneura stabulans. 

Calliphora erythrocephala. 

Palleuria rudis. 

Lucilia csesar. 

Lucilia regina. 

Sarcophaga ha?matoides. % 

Eristalis arbustorum. 

Anthomyia. 

Protozoa. — The rhizopoda are essentially characterized by the fact 
that locomotion does not take place by the aid of independent organs, 
but by means of pseudopodia, viz., protoplasmic processes which the 
animal is capable of protruding from any portion of its body. Six 
orders have been described by zoologists, but only one, or possibly 
two, have thus far been found in the feces. 

Whether or not representatives of the monera occur in the feces of 
man is still an open question. If so, they are apparently of no 
pathological significance. 1 

Of the amcebina, on the other hand, a most important member has 
been found, viz., the Amoeba coli (Losch). 

The history of the discovery of this parasite and its relation to 
those severe forms of tropical dysentery and liver-abscess which are 
met with also in our more temperate zones is of much interest, and 
at the same time illustrates the great importance which attaches to a 
systematic examination of the feces in all aggravated forms of diarrhoea. 

1 Nothnagel, loc. cit., p. 110. Grassi, cited by Bizzozero. v. Jaksch, Wien. klin. 
Woch., 1888, vol. i. p. 511. 



PATHOLOGY OF THE FECES. 



299 



Amoeba coli (Losch). — In 1875 Losch 1 discovered in the stools 
of dysenteric patients actively moving cell-like bodies of a roundish, 
pear-shaped, oval, or irregular form. He did not regard these as the 
cause of the disease, however, but looked upon them as only accident- 
ally present. Similar bodies were observed in Hong-Kong by Nor- 
mand in cases of colitis ; and also by v. Jaksch. Sansino found 
them in a case in Cairo, and Koch in East Indian dysentery. It is 
interesting to note that Koch was the first to suspect the existence 
of a definite relation between dysentery and these organisms. Cun- 
ningham claims to have found amoebae frequently in the stools of 
cholera patients at Calcutta, and Grassi in normal stools, but espe- 

Fig. 56. 




Amoeba coli. 

cially abundant in cases of chronic diarrhoea. Whether all these 
observations are correct, and whether the organisms observed were 
identical in all cases, is, of course, difficult to say. So much is cer- 
tain, that the subject was still a very unsettled one when Kartulis 2 
announced "that dysentery, and tropical liver-abscess associated 
with dysentery, are caused by the presence of the Amoeba coli," 
basing his conclusion upon an examination of five hundred cases. 
The fact that this parasite was absent in all other intestinal diseases, 
such as typhoid fever, intestinal tuberculosis, the ordinary forms of 
diarrhoea, etc., speaks strongly in favor of KartuhV view. 

In perfect accord with these observations are those made at the 
Johns Hopkins Hospital/ Osier 4 was the first in. this country to 

1 Loescb, " Massenbafte Entwickelung v. Amoben im Dickdarm," Vircbow's Archiv, 
vol. lvi. 

2 Kartulis, " Zur Aetiologie d. Dysenteric in Egypten," etc,, Vircbow's Arcbiv, 1885, 
vol. cv., and 1889, vol. cxviii. Centralbl. f. Bakt. u. Parasit.. 1890, vol. vii. 

3 Councilman and Larleur, " Amoebic Dysentery," Jobns Hopkins Hosp. Eep., 1891, 
vol. ii. C. E. Simon, Jobns Hopkins Hosp.' Bull., 1890. 

4 Osier, Jobns Hopkins Hosp. Bull., 1890. 



300 THE FECES. 

demonstrate the presence of the Amoeba coli in a case of liver- 
abscess, both in the pus of the abscess and in the stools. Stengel, 
Musser, Dock, and others confirmed these observations, so that the 
pathogenic character of the Amoeba coli may now be regarded as an 
established fact. 1 This statement is based not only upon the few 
facts, more historical in character than otherwise, which have just 
been detailed, but rather upon the ensemble of collected data, among 
which the absence of micro-organisms other than the amoeba in the 
pus of the liver-abscesses, and the constant presence of the latter 
in such cases, rank among the most important. It is to be noted, 
however, that different forms of tropical dysentery exist, and that 
the Amoeba coli is essentially associated with the more chronic form, 
while the acute types are probably of bacillary origin (see Shiga's 
bacillus). 

The size of the amoebae varies from 10 p. to 20 p.. When at rest 
their outline is, as a rule, circular, occasionally ovoid ; but when in 
motion they present the extremely irregular contour of moving 
amoeboid bodies (Fig. 56). The protoplasm can be differentiated 
into a translucent, homogeneous ectosarc or mobile portion, and a 
granular endosarc, containing the. nucleus, vacuoles, and granules. 
Within the endosarc the vacuoles constitute the most striking feat- 
ure. Sometimes the interior seems to be made up of a series of 
closely set clear vesicles of pretty uniform size. As a rule, one or 
two larger vacuoles are present, the edges of which are not infre- 
quently surrounded by fine dark granules. True contractile vesicles 
displaying rhythmic pulsations have not been observed, although the 
vacuoles may at times be seen to undergo changes in size. In some 
the nucleus is quite distinct, while in others it may be altogether 
invisible. The protoplasm of the amoebae is strongly basophilic. 
The organism can be cultivated artificially on hay-infusion in the 
presence of bacteria (Miller). 2 

Most distinctive are the movements of these bodies. From any 
part of the surface a rounded, hemispherical knob will project, and 
with a rapid movement the process extends and the granules in the 
interior flow toward it. In these movements the clear ectosarc 
seems to play the most important part. 

The Flagellata s. mastigophora differ from the rhizopoda in being 
provided with from one to eight flagella, which serve as organs of 
locomotion and possibly also for the apprehension of food-particles. 
Representatives of two orders only, viz., the monadina and isomasti- 
goda, have been found in the feces. Of the monadina in turn only- 
one family, viz., the cenomonadina, and of the isomastigoda only 
two families, the tetramitina and polymastigina, are represented. 3 

1 For the more recent literature see especially H. F. Harris, " Amoebic Dysentery," 
Am. Jour. Med. Sci., 1898, p. 384. 

2 C. O. Miller, " The Cultivation of Arnoebse," Contributions to the Science of Medi- 
cine, by the pupils of W. H. Welch, Baltimore, 1900, p. 511. 

3 W. Janowski, Zeit. f. klin. Med., vol. xxxi. p. 445. 



PATHOLOGY OF THE FECES. 301 

The cenomonadina are small, oval, frequently elongated bodies, 
provided with one long flagellum at the anterior end, at the base of 
which food-vacuoles are situated. At the posterior end amoeboid 
movements may be observed, and there can be no doubt that the 
taking up of food, to some extent at least, also occurs by the aid of 
pseudopodia. To this family belongs the cercomonas of Davaine 
and Lambl. The tetramitina are small, elongated bodies, provided 
with four flagella and a lateral, undulating membrane, which was 
formerly mistaken for a posteriorly directed flagellum. The tail- 
end of the organism tapers to a point. The nucleus is located at 
the base of the flagella. To this family belongs the parasite which 
was first discovered by Donne" in the vagina, and which later was 
found also in the feces, and which has been variously designated as 
Trichomonas hominis, Cercomonas coli hominis, etc. 

The polymastigina are small, somewhat oval bodies, provided with 
two or three flagella, situated either anteriorly or laterally — two or 
three on each side — while at the same time two additional flagella 
issue from the posterior end, which may either be rounded off or 
taper to a point. To this family belongs the Megastoma entericum 
of Grassi. 

Only three parasites belonging to the order of the flagellata have 
thus far been encountered in the human feces, viz., the Cercomonas 
hominis of Davaine and Lambl, the trichomonas of Donne, and the 
Megastoma entericum of Grassi. To judge from the earlier litera- 
ture upon the subject, many others have also been found, but more 
modern investigations have shown that they are in reality identical 
with the three just mentioned. The question whether or not these 
flagellate bodies are of pathological importance still remains sub 
judice. They are apparently met with only in diseases associated 
with diarrhoea, and it appears that in some cases at least this is 
directly dependent upon their presence ; in others the impression is 
gained as though they merely maintained an already existing diar- 
rhoea referable to other causes ; while in a third class of cases no 
relation can be discovered between their presence and the disease in 
question. Cohnheim 1 has recently pointed out that living infusoria 
in the feces may be a symptom of a primary chronic stomach affec- 
tion (gastritis, usually the atrophic form). According to the same 
writer, encysted infusoria may also be found in the feces of healthy 
individuals, but in such cases Ave may assume that at some time pre- 
viously a gastritis or a gastro-enteritis has existed. He thinks they 
have no pathogenic significance, and are merely of symptomatic- 
diagnostic interest. 

Cercomonas of Davaine-Lambl : syn., Cercomonas hominis (Da- 
vaine) ; monas (Marchand) ; Monas lens (Grassi) ; Monas mono- 
mitina (Grassi). The adult organism (see Fig. 57) is oval or 

1 P. Cohnheim, Deutsch. med. Woch., 1903, vol. xxix. p. 248. 



302 



THE FECES. 



roundish in form, and provided anteriorly with a single long flagel- 
lum and posteriorly with a tail-like appendage. Its length varies 
from 0.005 to 0.014 mm. The younger forms are pear- or S-shaped, 
and sometimes irregular in outline ; the flagellum is either absent 
or only rudimentary. 



Fig. 57. 




Cercomonas intestinalis. 
a, Cercomonas of Davaine, after Leuckart; b, Cercomonas intestinalis, after Lambl ; c, d, 
same, ordinary forms ; e,f, same, well-developed forms ; g, h, i, same, degeneration-forms ; k, I, 
same, abortive forms. 

Upon prolonged observation it will be seen that the adult parasite 
loses its flagellum and may protrude a protoplasmic process instead, 
while vacuolation occurs at the same time, indicating approaching 
death. 1 

Trichomonas, Donne : syn., Trichomonas vaginalis (Donn6) ; Tricho- 
monas hominis (Grassi) ; monocercomonas (Grassi) ; cimamomonas 
(Grassi) ; Protorycomyces coprinarius (Cunningham and Lewis) ; 
Cercomonas coli hominis (May) ; Trichomonas intestinalis (Leuckart 
and Roos) ; Cercomonas s. Bodo urinarius (Kunstler). The parasite 
(Fig. 58) is oval or spindle-shaped and measures from 0.012 to 0.03 
mm. in length by 0.01 to 0.015 mm. in breadth. From its anterior 
pole four flagella are given off, which are almost as long as the 
organism itself. From this point an undulating membrane extends 
laterally to the posterior pole, which may be rounded off or tapers to 
a tail-like appendage. This membrane is best seen when the move- 
ments of the flagella have ceased, as in specimens fixed in mercuric 
chloride solution (1 : 5000). The nucleus is situated at the base 

1 Lambl, Prag. Vierteljahr., 1859, vol. lxi. p. 1. Davaine, Traite des entozoaires, 
1860, Paris. Marchand, Virchow's Archiv, 1875, vol. lxiv. p. 293. Zunker, Deutsch. 
Arch. f. prakt. Med. ? 1878. 



PATHOLOGY OF THE FECES. 



303 



of the nagella, but is usually visible only in stained specimens 
(methyl ene-blue). At times the organisms may be observed to 



Fig. 58. 




Trichomonas intestinalis. 
a, a', c, trichomonas of the urine, after Marchand; b, Trichomonas vaginalis, after Donne; 
b', same, after Scanzoni and Kolliker; d, Trichomonas intestinalis, after Piccardi ; e,e?,e"> 
same, amoeboid forms; /,/, trichomonas of the urine, after Dock. 



assume an amoeboid form ; the movements of the nagella have then 
ceased, and pseudopodia-like processes are protruded. The parasite 
is identical with the trichomonas which has been found in the vagiua 
and in the urine. 1 When present in the feces the organism is usually 
seen in large numbers. Not infrequently it is found associated with 
other intestinal parasites. 

Megastoma entericum, Grassi : syn., Cercomonas intestinalis (Lambl); 
Megastoma intestinal e (Butschli); Lamblia intestinalis (Blanchard); 
Dimorphus muris (Grassi). The parasite (Fig. 59) is pear-shaped, 
and measures from 0.01 to 0.021 mm. in length by 0.0075 to 0.05 
mm. in breadth. In its anterior portion a more or less well-marked 
depression can be made out, which constitutes the peristome or 
mouth-opening of the organism. It is provided with eight nagella, 
grouped in pairs. The first pair originates on the sides of the peri- 
stome and is directed backward. The second and third pair are 
situated somewhat posteriorly and are likewise directed backward, 
while the fourth pair issues from the tapering tail-end of the body. 



1 Marchand, loc. cit. Zunker, loc. cit. 
Path, u. Therap.. 1894, vol, vi. 



p. 236. Hosier u, Peiper, Nothnagel's Spec. 



304 



THE FECES. 



In fresh specimens the eighth flagella can usually not be made out, 
as the third and fourth pair are frequently agglutinated. The best 
results are obtained when the organism has been killed with mercuric 
chloride solution. The individual flagella vary from 0.009 to 0.014 



Fig. 59. 




Megastoma entericum. 
a, b, b', c, c', c", c'", various forms of Cercomonas intestinalis, after Lambl ; d, d', encysted 
forms of Megastoma entericum, after Grassi and Schewiakoff; e, Megastoma entericum, 
adult form. 

mm. in length. In the anterior portion of the peristome two round, 
hyaline bodies can be recognized, which represent nuclei. Vacuoles 
are absent, and nutrition occurs through osmosis, the parasite adher- 
ing to epithelial cells by its peristome. When treated with fixing 
solutions the chitinous envelope can be readily recognized. In the 
encysted form the organism is oval and measures from 0.007 to 0.1 
mm. in diameter. 

Grassi observed the organism in mice, rats, cats, dogs, rabbits, and 
sheep. 1 

Balantidium coli, Stein : syn.. Paramecium coli (Malmsten). The 
organism is oval and measures from 70 // to 110 fi in length by 
60 fi to 72 fi in breadth. It is covered entirely with fine, actively 
motile cilia, which are grouped most densely about the funnel-shaped 
mouth, while at the anus only a few are seen. An ectosarc and an 
endosarc may be distinguished, and the parasite possesses the power 
1 Grassi U, Schewiakoif, Zeit. f, wia*. Zoologie, 1888, vol. xlvi. p. U3. 



PATHOLOGY OF THE FECES. 305 

to change its shape, and may appear quite round. In its interior 
we find a large, somewhat kidney-shaped nucleus, two contractile 
vesicles, and frequently fat-droplets, starch-granules, etc. 

The parasite is probably pathogenic, but comparatively uncommon 
outside of Sweden, Finland, and Russia. 90 cases have thus far 
been reported (1903). Of these, 25 occurred in Sweden, 14 in 
Finland, 24 in Russia, 11 in Germany, 5 in Italy, 1 in the Sunda 
Islands, 2 in the United States, 6 in Cochin China, 1 in Africa, and 
1 in the Philippines. Infection occurs through the dejecta of swine. 

Strong and Musgrave report that in their case blood examination 
showed a relative increase of the eosinophiles. 

From 200 to 300 organisms have been encountered in a single 
drop of the liquid feces. 1 

The fourth class of protozoa, viz., the Gregarina or sporozoa, 2 is 
also said to be represented in the human feces. The coccidia and 
psorosperms belong to this order. They are oval bodies, measuring 
about 0.022 mm. in length, and contain in their interior a large 
number of small nuclei arranged in groups. They are entirely 
devoid of organs of locomotion, and obtain their nutriment by 
endosmosis. Reproduction occurs in a common capsule, which 
bursts at a certain time and sends forth a whole generation of fully 
developed organisms. In human pathology they have become of 
interest in so far as certain observers have ascribed to them a role in 
the etiology of neoplasms. A disease of the liver analogous to the 
psorospermiasis of rabbits has also been described in man, and para- 
sites belonging to the same order have been observed in the skin. 

In this connection I wish to refer to the occurrence of Laveran's 
Plasmodium malarice enclosed in red corpuscles, in the stools of cases 
of malarial colitis. In one case of chronic malarial intoxication 
with dysenteric symptoms the diagnosis was first made after an 
examination of the stools for amoebae ; these were absent, however, 
while a number of plasmodia could be demonstrated, pointing to 
the probable nature of the colitis. 

Vermes. — The class vermes has interested physicians since time 
immemorial, and is referred to in the writings of Hippocrates and 
others, special mention being made of the ascarides, called lumbrices, 
and the platodes, called lati. Speaking of the former, Lucas Tozzi, 
in 1686, says : " They find their way into the heart and its pericar- 
dium, into the brain, the lungs, the vein's, and gall-bladder, where 
they are difficult to i catch.' " The same author, speaking of their 
effects upon the body, enumerates the following conditions as caused 
by their presence : epilepsy, vertigo, sopors, delirium, convulsions, 

1 Malmsten, Virchow's Archiv, 1897, vol. xii. p. 302. Sievers, " Ueber Balantidium 
Coli im menschlichen Darmkanal," Arch. f. Verdauungskrank., vol. v. p. 445. Ja- 
nowski, "Balantidium Coli," Zeit. f. klin. Med., vol. xxxii. p. 303. Henschen, Arch, 
f. Verdauungsk., 1901, vol. vii. p. 501. Solorojew, Centralbl. f. Bacter., 1901, vol. xxix. 
pp. 821 and 849. A. Ehrenrooth, Zeit f. klin. Med., 1903, vol. xlix. p. 321. 

2 v. Wasielewski, Sporozoenkunde, 1896. 

20 



306 



THE FECES. 



headache, syncope, palpitations, feelings of anxiety, cough, vomiting, 
nausea, diarrhoea, hiccough, prickling, borborygmi, erosions, tabes, 
acute and chronic fevers, and innumerable other maladies. 

It was even then deemed very important to make a diagnosis 
before the administration of an anthelmintic — a point which is well 



Fig. 60. 






Taenia saginata. 
a, natural size ; b, head much enlarged ; c, ova much enlarged. 

to bear in mind at the present day, and the eggs, segments, or para- 
sites themselves should be sought for in every suspected case before 
treatment is begun. 

Taenia saginata, Goeze : syn., T. mediocanellata (Kuchenmeister) ; 
T, incruris (Huber) ; T, dentata (Nicola). This parasite (Fig. 60) 



PATHOLOGY OF THE FECES. 307 

is the most common tapeworm both in Europe and North America. 
Infection occurs through the ingestion of measly beef. Its length 
varies from 4 to 8 m. The head, which is devoid of a rostelluru, 
is surrounded by four pigmented suckers, each of which is encircled 
by a dark line. The individual segments are quite thick and opaque, 
and diminish in length as the head is approached, the largest measur- 
ing from 2 to 3 cm. They are each provided with a very much 
branched uterus, which opens laterally, the primary branches num- 
bering about twenty on each side. The ova are elliptical in form, 
of a brown color, and usually enclosed in a distinct vitelline mem- 
brane. Upon careful observation a double contour with delicate 
radiating striae can be discerned. In the interior the embryos are 
seen imbedded in a brown, granular material. 

The larval form of Taenia saginata, the so-called Cysticercus 
taeniae saginatae (Leuckart), or the Cysticercus bovis (Cobbold), has 
been encountered in cattle, the Kocky Mountain "antelope," the 
llama, and the giraffe. In the human being it has not as yet been 
observed. 1 

Taenia solium, Kudolphi : syn., T. cucurbitina, plana, pellucita, 
Goeze. This parasite (Fig. 61) is far less common in this country than 

Fig. 61. 




Head of T. solium. X 45. (Leuckart.) 

the Taenia saginata, and may indeed be regarded as a curiosity. In 
Germany, also, it is only rarely met with now, while formerly it 
was the most common tapeworm in that country. This change is 
undoubtedly owing to the fact that raw pork is now eaten less fre- 
quently. In Asia and Africa it is more common. 

Taenia solium is usually much shorter than Taenia saginata, rarely 
exceeding 3.5 m. in length. Most characteristic is the head, which 
is ^provided with four pigmented suckers and a rostellum, furnished 
with from twenty-four to twenty-six hooklets arranged in a double 
row. The mature segments measure from 1 to 1.5 cm. in length 

1 J. Ch. Huber, Die Darmcestoden des Menschen. Bibliograph. d. klin. Helminthol., 
Heft 3 No. 4, p. 69, Miinchen, 1892. B. Leuckart, Die Parasiten des Menschen, 
etc., 2d ed., 1880, Pt. 1. 



308 THE FECES. 

by 6 to 7 mm. in breadth, and contain a uterus which has only five 
to seven branches, thus differing greatly from that of Taenia saginata. 
The ova are round, of a brownish color, and surrounded with a 
thick, radially striated membrane ; in their interior the hooklets 
of the embryos can usually be made out. 

The larval form of this tapeworm, the Cystieercus eettulosce, has 
been found in swine, the wild boar, in monkeys, in the brown bear, 
in the dog, etc. At times, though rarely, an auto-infection with the 
proglottides of Taenia solium has also been observed in the human 
being. Under such conditions the embryos of the worm are set free 
in the stomach, and may then migrate into various parts of the 
body, where they become encysted. Most commonly the cysticerci 
are found in the skin ; they have, however, also been observed in 
the heart, the lymph-glands, liver, bones, tongue, spinal canal, the 
brain, and the eyes. I have had occasion to observe a case of this 
kind at the Johns Hopkins Hospital (reported by Osier). The 
patient, a laboring-man, had never worked as a butcher or a cook, 
and never had a tapeworm. The cystieercus nodules, which were 
situated between the skin and the fascia, were very numerous, 
seventy-five being counted on one day. One of these nodules was 
removed for examination, and was shown to be referable to the cysti- 
eercus of Taenia solium. The only subjective complaints in this case 
Avere pains and stiffness in the arms and legs. The individual cys- 
tieercus was elliptical or roundish in form, measuring from 1 to 
10 mm. in diameter. In its interior the characteristic hooklets 
were seen. 1 

Taenia nana, v. Siebold : syn., hymenolepsis (Weinland). This 
parasite (Fig. 62) seems to be the most common tapeworm of Italy 
and Egypt. It has also been seen in Buenos- Ayres, in Bangkok, 
Siam, and a few isolated cases have been reported in England and 
in Germany. In the United States the parasite seems to be not at 
all uncommon, but has probably been overlooked in many cases. 
Stiles states that in his laboratory eighteen cases have been diag- 
nosed within a year (1902). It is found especially in young people, 
and often causes severe nervous symptoms, such as convulsions, 
loss of consciousness, and even melancholia. It is only 8 to 25 
mm. long and 0.5 mm. broad. The head is ball-shaped and pro- 
vided with four suckers and a rostellum, bearing twenty-four to 
twenty-eight hooklets arranged in a single row along its anterior 
edge. The individual segments are of a yellowish color and about 
four times as broad as long. The uterus is oblong and contains 
numerous ova, which are colorless, oval, and surrounded by a 
distinct non-striated membrane. They measure from 0.839 to 

1 Huber, loc. cit. Leuckart, loc. cit. ; and Blanchard, Traite de Zoologie medicale, 
vol. i., Paris. The Inspection of Meats for Parasites, Bull. No. 19, Bureau of Animal 
Industry, Washington, 1898. 



PATHOLOGY OF THE FECES. 



309 



0.060 mm. in size. In their interior the embryonic worm, pro- 
vided with five or six booklets, may be distinguished. The 
number of worms which may at times be found in the digestive 
tract is most astonishing, 5000 and even more having been counted 
on several occasions. The cysticercus stage occurs in snails, which 
are frequently eaten raw in Egypt and Italy. Taenia nana has been 
identified with the Taenia murina of rats and other rodents. 1 

Fig. 62. 






Taenia nana. Head, with rostellurn drawn in ; proglottis ; egg. (v. Jaksch.) 



Taenia diminuta, Rudolphi : syn., Taenia navapunctata (Weinland) ; 
Taenia minima (Grassi) ; Taenia varerina (Parona) ; Taenia lepto- 
cephala (Creplin). Taenia diminuta was first described in man by 
Leidy, Grassi, and Parona. It measures 20 to 60 mm. in length, 
and is armed with two suckers, but is without a rostellurn. The 
ova resemble those of Taenia solium. The cysticercus occurs in 
certain caterpillars and cocoons. In man it has been found in only 
six instances. 2 

Dipylidium caninum, Linne : syn., Taenia canina (Linn6) ; Taenia 
moniliformis (Pallas) ; Taenia cucumerina (Bloch) ; Taenia elliptica 
(Batsch). The parasite is found almost exclusively in children, 
infection occurring through dogs and cats. In the United States 
the disease is apparently rare. The only case reported is that of 
Stiles. 3 The larval form is found in lice and fleas. The worm itself 
measures from 15 to 35 cm. in length. The head is small, globular; 
the rostellurn club-shaped with 3 or 4 transverse rows of hooks (about 
60 in number) of rose-thorn form; anterior hooks 15 fi, posterior 
hooks 6 /a ; suckers relatively large, rather elliptical. Segments 80— 
120 in number; gravid segments 8-11 mm. long, 1.5-3 mm. broad ; 
often reddish-brown in color. Genital pores at equator or in posterior 

1 Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. i. p. 97. Grassi u. Calandruccio, 
Ibid., 1887, vol. ii. p. 282. Comini, Ibid., p. 27. Bilharz, cited by Leuckart. C. W. 
Stiles, New York Med. Jour., Nov. 7. 1903. 

2 Leidy and Parona, cited bv Leuckart. 

3 C. W. Stiles, Arner. Med., 1902, vol. v. p. 65. 



310 



THE FECES. 



half of segment ; uterus forms egg capsules, each containing from 8 
to 20 eggs, eggs globular, 43-50 p. in diameter. The ova contain 
embryos already armed with hooklets (Stiles). In diagnosis Stiles 
suggests that search be made in the feces for the peculiar elongated 
elliptical tapeworm segments (Fig. 63). Microscopical examination 
of the feces for eggs is less certain than in cases of infection with 
Taenia saginata, Taenia solium, or Dibothriocephalus latus, since 
Dipylidium is much smaller and less prolific than any of these three 
forms. 1 

Fig. 63. 




a, Dipylidium caninum (taken from Stiles) ; b, gravid segment (after Diamare) ; c, head, 
showing four rows of rose-thorn hooks on the rostellum and four unarmed suckers (Stiles) ; 
d, egg, showing six hooks of the embryo (Stiles). 



Taenia africana, v. Linstow. 2 This parasite has been found in two 
instances, in the case of two native soldiers at Nyasa Lake. Like 
the scolex of Taenia saginata that of the present species is devoid of 
hooklets. Its length is about 1.4 m. ; the number of segments 
about 600. They are all much broader than long. The uterus 

1 A. Hoffmann, Jahresb. f. Kinderheilk., 1887, vol. xxvi. Hefte 3 u. 4. Kriiger, St. 
Petersburg, med. Woch., 1887, vol. xii. p. 341. Brandt. Centralbl. f. Bakt. u. Parasit., 
vol. v. p. 99. 
v. Linstow, Centralbl. f. Bakt. u. Parasit., 1900, vol. xxviii. p. 485. 



PATHOLOGY OF THE FECES. 



311 



consists of a main portion running fore and aft, from which from 
15 to 24 side branches issue, which do not branch dichotomously 
and are so closely packed that they cannot be recognized with the 
naked eye. 

Taenia Madagascariensis (Grenet). — This parasite has been found in 
Madagascar, in Mauritius, in Bangkok, and in a Demarara Indian. 
The worm attains a length of from 25 to 30 cm. and is composed of 
from 500 to 600 trapezoid segments. The rostellum is surrounded by 
a double row of minute hooklets. The suckers are round and quite 
large. Blanchard suggests that the cockroach may be its interme- 
diary host. 

Bothriocephalus latus, Bremser : syn., Tsenia lata (Linn6) ; Diboth- 
rium latum (Rudophi) (Figs. 64-67). This worm is usually 
5-10 m. long and of a reddish-gray color. Longer specimens, 
however, may also be encountered. In Wilson's case 82 feet of 
segments were obtained from two worms, so that the length of each, 

Ftg. 64. 




Bothriocephalus latus : A, B, twin segments. (Wilson.) 



supposing both to have been of the same size, must have been more 
than 40 feet. The head is almond-shaped and upon its flat surfaces 
two distinct grooves can be discerned, which probably act as suckers. 
It measures 2—3 mm. in length by 1 mm. in breadth. The neck is 
very short and passes at once into the body segments. Adjacent 
segments can often be distinguished only by means of the recurrence 
of the sexual apparatus, which appears regularly in spite of the 
imperfect individualization of the segments. The ripe segments are 
almost square in form, with the genital apparatus opening in the 
median line. The fully developed segments measure 2.5—4.5 mm. 
in length by 8-14 mm. in breadth. The total number of segments 
may far exceed 3000. The frequent occurrence of imperfect and 
abortive types of twin segments may be considered an almost dis- 
tinctive feature of the bothriocephalus family (Wilson). The uterus 
presents 4 to 6 convolutions on each side, which become especially 
distinct when the segments are placed in water or are exposed to 
the air. A rosette-like appearance is then noted, which is quite 
characteristic. The rosette deepens in color in proportion to the 
number of ova which the uterus contains, and toward the tail of the 
parasite, from the segments of which many or all the eggs have 



312 THE FECES. 

been discharged, the rosette tends to become light in color, and may 
indeed appear whiter than the surrounding parenchyma. The eggs 
(Fig. 65) are oval, 0.06—0.07 mm. long and about 0.045 mm. broad ; 
they are enclosed in a brown envelope, at the anterior end of which 
a little lid can be recognized. Their contents consist of protoplasmic 
spherules, all of about the same size, which are lighter in color in 
the centre than at the periphery. In infested individuals they are 
constantly found in the stools. 

The larva? have been found in various fresh-water fishes, such as 
the perch, the ling, the turbot, in various members of the trout 
family, but they are most commonly encountered in the pike. It is 
thus readily understood why the parasite is most common in lake 
regions, as in Switzerland, northern Russia, southern Scandinavia, 

Fig. 65. Fig. 67. 




Fig. 66. 




Plerocercoid. (Braun.) Embryo with cilia and hooklets : A, bundles of muscle- 
fibres. (Leuckart and Braun.) 

and northern Italy. It is seldom seen in middle Germany, but is 
so common in Ireland that Cobbold named it the Irish tapeworm. 
Outside of Europe it is most common in Japan. In the United 
States a few imported cases have been observed by Walker and 
Leidy, Packard, Hageestam, Riesman, Stengel, McFarland, and 
Wilson. 

Multiple infection has been repeatedly observed. Bottcher notes 
a case in which 100 worms were found ; Roux and Eichhorst both 
speak of cases with 90, Heller of one with 38, and in Wilson's case 
2 were undoubtedly present. When more than one occurs, the 
growth of the individual is impeded, and small specimens are then 
usually seen (3-5 feet or more). Clinically the parasite is of especial 
interest as its presence in a certain percentage of cases is associated 
with the clinical picture of a pernicious anaemia ; in others, how- 
ever, no deleterious eifect upon the red corpuscles is noted, although 
several worms may be present in the intestinal tract. 



PATHOLOGY OF THE FECES. 



313 



Besides in man, the worm has been encountered in the dog, cat, 
the seal, and in some water birds. The ovum after being discharged 
in the feces, during a variable period of incubation in the water 
develops into the onchosphsera, a ciliated larva w T ith 6 hooklets 
(Fig. 67). The larva is then liberated from the ovum by passing 
through the lidded end, and by means of its cilia moves rapidly 
through the w^ater. If not eaten by fish, it dies ; otherwise it 
develops into the bothriocephalus measle, the plerocercoid (Fig. 66), 
which has both head and tail. Infection of man then occurs when 
such fish are eaten either raw or but partly cooked. In man the 
cysticercus stage has not been observed. 1 

Krabbea grandis, Blanchard. This parasite has been observed in 
only one instance — in Japan. It is said to resemble certain bothrio- 
cephali which are found in seals. The genital organs are double in 
each segment. The vulva and uterus open ventrally. The worm 
attains a length of 10 m. with a breadth of 2 cm. 

Trematodes. — The various forms of distoma which belong to this 
order are essentially hepatic parasites, and rarely occur in the feces. 

Distoma hepaticum, Abildgaard : syn. t Fasciola hepatica (Linne) 
(Fig. 68). This, the most common liver-fluke, is 28 mm. long and 



Fig. 68. 



Fig. 69. 






Distoma hepaticum. (Leuckart. 



Distoma lanceolatum (x 8) and eggs. (v. Jaksch.) 



12 mm. broad; it is formed like a leaf. The leaf is provided with 
a sucker, and a second sucker may be found at its ventral surface. 
Between the two the genital opening is located, leading into a skein- 
shaped uterus. The eggs are oval, measuring 0.13 mm. in length 
and 0.08 mm. in breadth, the anterior end being provided with a lid ; 
their color is brown. In the United States the organism is practi- 

1 Schaumann, Zur Kenntniss d. sogenannten Bothriocephalus-Anaemie, Berlin, 
1894. Schauman u. Tallqvist, " Ueber d. blutkorperchenauflosenden Eigenschaften 
d. breiten Bandwurms," Deutsch. med. Wocb., 1898, p. 312. Kuneberg, Deutscb. 
Arch. f. klin. Med., 1887, vol. xli. p. 304. Askanazv, Zeit. f. klin. Med., 1895, vol. 
xxvii. p. 492. R. N. Wilson, " Bothriocephalus, Report of a Case of Double Infection." 
Am. Jour. Med. Sci., 1902, vol. cxxiv. p. 262. 



314 THE FECES. 

cally unknown, while in Germany it is most common in sheep. In 
the human being it is rare in both countries. It occurs in cattle, 
sheep, swine, cats, rabbits, etc. Infection occurs through a small 
snail, the Linnaeus minutus, which is found, in Germany especially, 
upon watercress. 1 

Distoma lanceolatum, Mehlis, has been found in only five cases, 
all of which occurred in Germany (Fig. 69). It is much smaller 
than Distoma hepaticum, measuring 8 to 9 mm. in length, by 2 to 
3.3 mm. in breadth. It is lancet-shaped, tapering toward the head- 
end, but otherwise closely resembles the above parasite. The ova 
are 0.04 mm. long, 0.03 mm. broad, and contain fully developed 
embryos. In cattle, sheep, and hogs the organism is quite common. 2 

Distoma Buski, Lancaster : syn., Distoma rhatonisii (Poirier) ; 
Distoma cranum (Busk). The parasite has been observed in China, 
Sumatra, the Straits Settlements, Assam, and India. It is the largest 
distoma occurring in man, measuring over an inch in length. It prob- 
ably inhabits the upper portion of the intestine and may give rise to 
attacks of recurring diarrhoea and other signs of intestinal irritation. 
Infection probably occurs through certain fishes and oysters. 3 

Distoma sibiricum, Winigradoff: syn., Distoma felinum (Rivolta). 
This parasite was found in Tomsk, by Winigradoff, in eight autop- 
sies out of one hundred and twenty-four. Askanazy also reports 
two cases of infection from eastern Prussia, in which the eggs were 
found in the stools. In one of the cases, which came to section, 
more than one hundred organisms were found in the biliary passages. 
Its length may reach 13 mm. The ova are 0.026 to 0.038 mm. long 
and 0.010 to 0.022 mm. broad. The intestine is simple and extends 
to the posterior extremity of the body. Its surface is smooth. 4 

Distoma spatulatum, Leuckart : syn., Distoma sinense (Cobbold) ; 
Distoma endemicum (Balz) ; Distoma japonicum (Blanchard). It 
has been observed in India, Mauritius, Corea, Formosa, China, 
Tonkin, and Japan, and it appears that in the two last-named 
countries it is quite common. It inhabits the biliary passages and 
gall-bladder. It is distinctly pathogenic. The ova may be found 
in the stools. The parasite possibly also occurs in cats. The 
intermediary host is not definitely known ; it may be some fresh- 
water mollusc. It is about 11.75 mm. long and 2 to 2.75 mm. 
broad. The living parasite is of a reddish color and translucent, so 
that it is possible to distinguish all its interior organs. The ova 
measure 0.028 to 0.030 mm. in length by 0.016 to 0.017 mm. in 
breadth, and are enclosed in a colorless envelope. 5 

1 C. W. Stiles, Jour. Comp. Med. and Vet. Arch., 1894, vol. sv., and 1895, vol. xvi. 
Huber, Trematoden. Bibliog. d. klin. Helminthol., Hefte 7 u. 8, p. 283. 

2 Leuckart, loc. cit., p. 137. 

3 Poirier, Centralbl. f. Bakt. u. Parasit., 1888, vol. ii. p. 186. 

4 Winigradoff, cited by Braun, Centralbl. f. Bakt. u. Parasit., 1894, vol. xv. p. 602. 

5 Blanchard, loc. cit. 



PATHOLOGY OF THE FECES. 



315 



Other parasites belonging to this order are Distoma conjunctum 
(Cobbold), Distoma heterophyes (v. Siebold), and Amphistomum 
hominis (Lewis and MacConell). The last-named appears to be 
common in elephants and has been encountered in natives of Assam, 
in two Indians in Calcutta, and in an East Indian immigrant in 
British Guiana. It is quite small, measuring from 5 to 8 mm. in 
length by 3 to 4 mm. in breadth and is characterized by the large 
size of its posterior suckers. 

Distoma heterophyes is the smallest distoma, so far as we know, 
which is found in man. It occurs in Egypt and is thought to be 
innocuous. 

Distoma conjunctum was discovered in an East Indian. Its 
surface is covered with minute spicules. It is not of much patho- 
logical importance. 

Fig. 70. 




Ascaris lumbricoides. (v. Jaksch.) 

o, worm, half natural size ; b, head slightly magnified ; c, eggs. (Eye-piece I., objective 8 A, 

Reichert.) 



Distoma haematobium and Distoma pulmonale are described in the 
sections on the Blood and the Sputum, respectively. 

Annelides. — The aunelides are very common intestinal parasites, 
and of these especially the nematodes. 

Ascaris lumbricoides, Linn6 (Fig. 70), is the cylindrical^ shaped 
worm so commonly seen in children and in the insane. The head 
consists of three projections or lips, which are provided with suckers 
and fine teeth. The male measures about 215 mm., the female about 
400 mm. in length. The tail-end of the male is rolled up on its 



316 



THE FECES. 



ventral surface like a hook, and is provided with papillae. The gen- 
ital aperture of the female is situated directly behind the anterior 
third of the body. The eggs are yellowish brown in color, almost 
round, and measure 0.06 mm. by 0.07 mm. in size ; they are sur- 
rounded by an irregular albuminous envelope, which is covered by 
a tough shell ; the contents are coarsely granular. 

Ascaris lumbricoides is found in all countries, and also infests the 
pig and the ox. Its presence may occasion severe nervous symp- 
toms, but fortunately this is rarely the case. 1 

Ascaris mystax, Zeder : syn., Ascaris marginata (Rudolphi) ; 
Ascaris alata (Bellingham) (Fig. 71). This worm is smaller and 
thinner than Ascaris lumbricoides, but otherwise very similar. The 
head is pointed and provided with wing-like projections, which con- 
stitute the main point of difference between the two. The male 
measures 45 to 60 mm. in length, the female 110 to 120 mm. Its 
ova are round, larger than those of Ascaris lumbricoides, and en- 
closed in a membrane which is covered with numerous small depres- 
sions. The worm is common in dogs and cats, but very rare in 
man. 2 

Ascaris maritima, Leuckart, also belongs to this class. It has 
been observed in only one case — in Greenland. 

Oxyuris vermicularis, Bremser : syn., Ascaris vermicularis (Linne) ; 
Ascaris grsecorum (Pallas) (Fig. 
72). The male is 4 mm., the 
female 10 mm. long. At the head 

Fig. 71. 



Fig. 72. 





Ascaris mystax. (v. Jaksch.) 
a, male ; b, female ; c, head ; d, egg. 



Oxyuris vermicularis. (v. Jaksch.) 
a, head ; b, male ; c, female ; d, eggs. 



three lip-like projections with lateral cuticular thickenings may be 
seen. The tail of the male is provided with six pairs of papillae, and 

1 Lutz, Centralbl. f. Bakt. u. Parasit, 1888, vol. iii. pp. 553, 584, 616. Hogg, Brit. Med. 
Jour., 1888, p. 121. Kartulis, Centralbl. f. Bakt. u. Parasit., vol. 1. p. 65. 

2 K. A. Rudolphi, Arch. f. Zool. u. Zoot., 1803, vol. iii. Pt. 2, p. 1. Idem, Entozoorum 
s. vermium intestinal, historia naturalis, Amstelaedami, ii. 2. 



PATHOLOGY OF THE FECES. 



317 



the female with two uteri. The eggs are 0.05 by 0.02 to 0.03 mm. in 
size, and covered with a membrane showing a double or triple contour ; 
in the interior, which is coarsely granular, the embryos are contained. 

The female worm lives in the caecum, but after impregnation 
travels downward to the rectum. Here it causes most annoying 
symptoms, which are especially distressing at night, when the organ- 
ism emerges from the anus. In doubtful cases of pruritus ani et 
vulvae an examination of the feces should be made for this parasite. 
The ova themselves do not occur in the feces. 1 

Anchylostomum duodenale (Dubini) : syn., Anchylostoma duo- 
denale (Dubini) ; Strongylus quadridentatus (v. Siebold) , Doch- 
mius anchylostomum (Molin) ; Sclerastoma duodenale (Cobbold) ; 
Strongylus duodenalis (Schneider) ; Dochmius duodenale (Leuckart) ; 
Uncinaria duodenalis (Roilliet) (Fig. 73). This organism belongs 

Fig. 73. 




Anchylostomum duodenale. (v. Jaksch.) 

a, male, natural size ; b, female, natural size : c, male, magnified ; d, female, magnified; 

e, head (eye-piece II., objective C, Zeiss) ; /, eggs. 

to the family Strongyloides, and is one of the most dangerous para- 
sites met with in the human being. It has been found in Italy, 
Germany, Switzerland, Belgium, Egypt, and in the West Indies 
(Jamaica). In the United States a few imported cases of anchylo- 
stomiasis have also been met with, but the impression has been 
general that the disease was quite rare. C. W. Stiles, however, 
has recently shown that a distinct species of the hook-worm exists 
in this country as also in the West Indies, viz., in Cuba and Porto 
Rico, the Uncinaria Americana, and that in the sand regions of 
the South infection with this parasite is common, especially among 
the lower classes of negroes in many of whom infection probably 
occurs in consequence of the habit of dirt-eating. 

1 Lutz, loc. cit. 



318 



THE FECES. 



From a pathological standpoint the parasite is of special interest, 
as its presence may give rise to severe and often fatal anaemia. Grie- 
singer was the first to point out that the so-called Egyptian chlorosis 
is produced by this organism. Subsequently it was shown that the 
same parasite was responsible for the anaemia which developed 
among the workers on the St. Gotthard tunnel, and which was 
common among brickmakers in certain districts in Germany. In 
this country the anaemia of the dirt-eaters has long been known in 
the South, and has been generally attributed to the peculiar habit. 
Its real cause is now manifest. While infection no doubt generally 
takes place by a direct transference of the embryos with dirty hands, 
it may certainly also occur through contaminanted drinking-water. 

Outside of man the parasite is not uncommon in dogs, cattle, and 
sheep. 

Fig. 74. 




Eggs of Unciuaria americana, in different stages of development (personal observation). 

Magnified about 300. 



The male is 6 to 11.5 mm. long, the female 10 to 18 mm. The 
head, which tapers somewhat, is turned toward the back ; the mouth 
capsule is hollowed out arid surrounded by 4 teeth ; 1 the tail of the 
male forms a 3-lobed bursa, while that of the female tapers coni- 
cally ; the genital opening is behind the middle of the body. Its 
eggs have an oval form and a smooth surface, measuring from 
0.05 to 0.06 by 0.03 to 0.04 mm. In their interior two or three 
segmenting bodies are found, which rapidly develop outside of the 
human body, so that after twenty-four to forty-eight hours embryos 
may be found in the same feces in which the eggs were observed, 

1 The American species has only one dorsal, conical tooth, which projects promi- 
nently into the buccal cavity (Stiles). 



PATHOLOGY OF THE FECES 



319 



or fully developed ova may be found after allowing the feces to 
stand for only a few hours (Fig. 74). When allowed to dry, the 
young parasites become encysted, but after remaining so even for 
from one to two weeks they are capable of infection. A second 
host for its cycle of development is, according to Leichtenstern, not 



necessary. 



Fig. 75. 



£V 



- 






- 




: 







Trichina spiralis in muscle. 



The habitat of the adult worm is the jejunum. It is rarely found 
in the feces. Its eggs, however, are common, and should be looked 
for in every case of anaemia the cause of which is not manifest, 
especially in miners, tunnel-workers, brickmakers, dirt-eaters, etc. 
Any specimen of fecal material will answer as a rule, but it is best- 
to procure a thin stool, as after a purge. It is then merely neces- 

1 Leichtenstern, Centralbl. f. klin. Med., 1885, vol. vi. p. 195 ; Deutsch. rned. Woch., 
1885, vol. xi. ; 1886, vol. xii. ; 1887, vol. xiii. Lutz, Volkmann's Sammlung, 1885, 
Nos. 255 and 256. American cases : C. W. Stiles, " The Significance of the Becent 
American Cases of Hook-worm Disease," 18th Annual Eeport Bureau Animal Indus- 
try, 1901. H. F. Harris, Amer. Med., Nov. 15, 1902, p. 776. A. J. Smith, Am. Jour. 
Med. Set, 1903, vol. exxvi. p. 768. C. F. Craig, Ibid., p. 798. 



320 THE FECES. 

sary to mount a small drop on a slide and to examine the covered 
specimen with a low power ; a Bausch & Lomb f is quite sufficient. 
A mental picture of the size of the eggs should be made, for I have 
known it to occur that an observer saw the eggs, but did not recog- 
nize them as such. Once seen, they are easily recognized again. 

Trichocephalus hominis, Schwank : syn., Trichocephalus dispar 
(Rudolphi) ; mastigodes (Zeder) ; trichuris (Buttner). This parasite, 
which belongs to the family Trichotrachelides, is formed like a whip, 
the last-end being the head-end, while the tail-end is very much 
thicker. The male measures 46 mm. and the female 50 mm. in 
length. The eggs are brownish in color, measuring 0.05 by 0.06 
mm. in size, and present a doubly contoured shell, with a de- 
pression at each end, closed by a lid. The contents are coarsely 
granular. The organism is said to be the most widely distributed 
intestinal parasite, occurring in Europe, North America, Asia, 
Africa, and Australia. Its habitat is the caecum. The living worm 
is only rarely found in the feces. 1 

Trichina spiralis (Owen) (Fig. 75) is rarely found in the feces. 
The male measures 1.5 mm. in length, and is provided with four 
papillae between the conical lips. The female is 3 mm. long. The 
uterus is situated nearer the head than the ovary, which opens into 
it. Fertilization occurs in the intestinal canal. The eggs develop 
into embyros in the uterus, emerge from this, and penetrate the 
intestinal walls, whence they are carried by the blood-current to the 
muscles. Trichinosis is far less common in the United States than 
in Europe. 2 The diagnosis of sporadic cases has been greatly facili- 
tated by the discovery of Brown that eosinophilia, often of high 
grade, is practically of constant occurrence during the acute stage 
of the disease (see page 102). 

Strongyloides intestinalis (Bavay) : syn., Anguillula intestinalis 
(Bavay) ; Anguillula stercorals (Bavay) ; Rhabditis stercoralis 
(Bavay) ; Leptodera stercoralis (Bavay, Cobbold), Leptodera intes- 
tinalis (Bavay, Cobbold) ; Strongyloides intestinalis (Bavay, Grassi) ; 
Pseudo-rhabditis stercoralis (Bavay, Perroncito) ; Rhabdonema 
strongyloides (Leuckhart) ; Rhabdonema intestinale (Bavay, 
Blanchard). 

In the feces of patients infested with the parasite in question the 
eggs of the mother-worm are only rarely found, and the worm itself 
probably never appears unless an anthelmintic has been administered 
and active catharsis established. Instead we find embryos (rhab- 
ditic form) measuring about 0.33 by 0.022 mm. in size. If the 
stools are kept, uncovered, at a temperature of about 37° C, their 
larvae undergo development and reach full growth and sexual dif- 
ferentiation in almost five days. The length of the full-grown 
female is about 1 mm. ; its breadth about 0.04 mm. The body is 

1 Ermi, Berlin, klin. Woch., 1886, vol. xxiii. p. 614. 2 Leuckart, loc. cit. 



PATHOLOGY OF THE FECES. 321 

cylindrical, slightly diminishing in size anteriorly and tapering to 
a sharp point posteriorly. When the worm retracts forcibly, slight 
transverse furrows may be seen. The mouth possessed dis- 
tinct lips and is continnous with a triangular oesophagus, which 
beyond a constriction dilates again into a second ovoid enlarge- 
ment. The intestine which follows ends in a little protrusion 
on one side of the body near the base of the tail. A little below 
the middle of the body, and on the ventral side, is the vulva, 
which leads to ,the uterus, extending from the intestinal ventricle 
to a point near the anus. Here the eggs may be massed in varying 
numbers. Sometimes the young have actually broken the shell of 
their eggs and may be seen free in the uterus ; but more commonly 
the ova, on deposition, contain well- formed motile embryos (filari- 
form brood). The male is about one-fifth smaller than the female. 
The testicle ends at the base of the tail in two small horn-like spic- 
ules with tapering ends, which are curved inward. These spicules 
contain canals ; they are of equal size and situated symmetrically on 
a transverse plan. The tail is coiled in the same direction as the 
spicules, and is half as long as that of the female. 

The sexually mature and differentiated forms just described repre- 
sent the Anguillula stercoralis of Bavay. They represent an inter- 
mediate generation, developing outside of the body, which forms a 
link in the chain of development of the mother-worm, the Anguil- 
lula intestinalis (Leuckart). 

Ordinarily infection takes place through the larvae of the sexually 
differentiated form. These filariform embryos are longer than the 
rhabditiform brood of Anguillula intestinalis (Fig. 76). They are 
provided with a cylindrical oesophagus descending down to about 
the middle of the body, and a tail, which instead of terminating in a 
fine point, is apparently truncated at its extremity. On maturation 
they give rise to the Anguillula intestinalis, which is encountered 
throughout the upper gastro-intestinal tract, especially in the lower 
part of the duodenum and the upper part of the jejunum, though 
occasionally they have also been found throughout the entire jeju- 
num and in the upper part of the ileum. On several occasions they 
have been found in the stomach. 

Anguillula intestinalis, viz., the parasitic mother-worm, is, accord- 
ing to Rovelli, parthenogenetic, while Leuckhart expressed the 
opinion that it might be hermaphroditic. Its length is abont 2.20 
mm., and its average breadth 0.03 mm. The body tapers a little 
anteriorly, and terminates posteriorly in a conical tail, the extremity 
of which is appreciably rounded and even a trifle dilated. The 
mouth is without horny armature, and shows 3 small lips. It opens 
into a cylindrical oesophagus, which occupies about one-fourth of 
the length of the animal, and shows neither swellings nor striations. 
The intestine extends nearly to the posterior extremity of the body, 

21 



322 



THE FECES. 



but is almost invisible in the middle part owing to the presence of 
a large elongated ovary. The vulva is situated in the posterior 
third of the animal, and the uterus contains usually 5 or 6 rather 
elongated ova. The anus is situated toward the base of the tail. 
The eggs are of a yellowish-green color, rather opaque, and appar- 
ently finely granular (Bavay) ; in their general appearance they 
resemble those of the Uncinaria (Fig. 76, A.). 

Fig. 76. 




A, egg of Strongyloides intestinalis (parasitic mother worm) ; B, rhabditiform embryo ; 
C, filariform embryo, derived by direct transformation, from a rhabditiform embryo. (Taken 
from Thayer.) 

While infection originally takes place through the filariform larvae 
of Anguillula stercoralis, an auto-infection with the larvae may also 
occur without the intervention of the sexually differentiated forms, 
by a direct transformation from the rhabditiform embryos of the 
parasitic mother-animal, and there is evidence to show that this 
latter cycle is indeed the more common. There is no evidence to 
show that the sexually mature intermediate generation ever develops 
in the intestinal tract during life. 



PATHOLOGY OF THE FECES. 323 

The time elapsing between infection with the filariform larvae 
and the appearance of rhabditiform embryos in the stools is about 
seventeen days. 

The parasite is the recognized cause of the so-called Cochin-China 
diarrhoea, and is of further interest from its resemblance to Anchy- 
lostoma duodenale, with which it is not infrequently found asso- 
ciated Excepting in very rare instances, it does not cause intestinal 
ulceration, and it is supposed that the injurious effects of the para- 
site are purely mechanical. It is possible, however, that these may 
also be owing to the irritating action of its excretory products. 
The clinical manifestations of the disease are mainly those of a 
chronic diarrhoea and a comparatively mild anaemia. There are 
usually 3 or 4 pasty stools a day. 

The organism was first discovered in individuals who had con- 
tracted severe diarrhoea in Cochin-China. Grassi and Parona later 
found the worm in Italy, and at the building of the St. Gotthard 
tunnel it was frequently seen in association with the Anchylostoma. 
Thayer was the first to find it in the United States, and it is in- 
teresting to note that two of his three cases must have become in- 
fested in either Maryland or Virginia. The third case may have 
originated in Austria ; in it the anguillula was associated with 
amoebae and the Trichomonas intestinalis ; it ended fatally, being 
complicated by liver abscess. Since then additional cases have been 
reported in the United States by Moore and Price. 

Other cases have been observed in Belgium, Holland, Martinique, 
Brazil, Sicily, the Dutch Indies, Egypt, Germany, Spain, and the 
Philippine Islands. 

Literature.— Grassi, Centralbl. f. Bakt. u. Parasit., 1887, vol. ii. p. 413. Leich- 
tenstern, Deutsch. med. Woch., 1898, p. 118. Perroncito, Arch. p. 1. sci. med., 1881, 
No. 2. Compt. rend, de 1' acad. des sci., 1882, No. 1. Teissier, Ibid., vol. cxxi. p. 171. 
Bavay, Ibid., 1876, vol. lxxxiii. p. 694 : Ibid., 1877, vol. lxxxiv. p. 266. Normand, 
Ibid., 1876, p. 316. W. S. Thayer Jour.' of Exper. Med., 1901, vol. vi. No. 1 (full liter- 
ature to 1901). M. L. Price, Jour. Am. Med. Assoc, Sept. 12, 1903 (literature to date 
since Thayer's paper). 

Insecta. — As the larvae of the various insects met with in the 
feces have been very little studied, they will not be considered at 
this place ; they are apparently of no clinical importance. 

Vegetable Parasites. — Among the pathogenic vegetable parasites 
the bacillus of cholera, of typhoid fever, and of tuberculosis, as well 
as the bacilli of Booker, the Bacillus coli communis, the Bacillus pyo- 
cyaneus, the Bacillus lactis aerogenes, the bacillus of Shiga, and the 
Proteus vulgaris, deserve especial consideration. 

The Comma-bacillus. — As early as 1848 certain "vibrios" were 
observed in abundance in the stools of cholera patients by Yirchow, 
and in 1849 by Pouchet, Britton, and Swayne, no importance, how- 
ever, being attached to their presence at the time. 



324 THE FECES. 

The first accurate and detailed studies of the organism found in 
cholera stools were made in 1883 by the members of the French 
and German commissions sent to Egypt to investigate the nature 
of the dreaded disease. The results of their work were first pub- 
lished by Koch in his report to the Berlin Sanitary Office in 1883, 
and in 1884 by Strauss, Poux, Nocard, and Thuillier. 

The clinical recognition of cholera Asiatica has now become a 
simple matter since Pfeiffer has demonstrated that the blood-serum 
of cholera patients possesses the property of causing arrest of 
motility and agglutination of the specific bacilli. Ordinary bouillon- 
cultures, however, can usually not be employed, as particles of the 
film when broken up may easily be mistaken for agglutinated 
bacilli. It is best in every case to make use of agar-cultures 
sixteen to twenty-four hours old, and to prepare emulsions in 
bouillon or normal salt solution as occasion requires. The emul- 
sion, moreover, should always be examined microscopically before 
use, so as to insure the absence of any conglomerations of bacilli. 
The blood is then diluted in the proportion of 1 : 10 or 1 : 15. If the 
test-tube method is employed, the tubes should be kept in the incubator 
(37° C.) for only one or two hours. Upon the slide the reaction is 
obtained in from five to twenty minutes. If no distinct agglutination 
is observed at the end of one hour, the diagnosis of cholera is rendered 
improbable. Dried blood retains its agglutinating properties for a 
considerable length of time, and may also be used for examination. 

The comma-bacillus is a slightly arched or half-moon-shaped 
little rod, and is somewhat shorter than the tubercle bacillus (Plate 
XV., Fig. 1 ). Occasionally two are placed end to end with their 
convexities in opposite directions, thus presenting the appearance 
of the letter S. They are provided with flagella. Koch detected 
these bacilli in the intestinal contents and feces, but rarely in the 
vomited matter, in Asiatic cholera only. In the stools they at times 
occur in such numbers as to constitute pure cultures. In plate- 
cultures kept at a temperature of 22° C. white colonies with serrated 
borders may be observed after twenty-four hours. The color of 
such a colony is slightly yellow or rose red, its central portion gradu- 
ally assuming a deeper tint, and finally becoming liquefied. Upon 
agar-plates the bacilli form a grayish-yellow, irregular, slimy coat- 
ing, but do not liquefy the culture-medium. In stab-cultures, after 
twenty-four hours, a whitish color may be observed along the line 
of the stab ; around this there is formed a funnel-shaped depression, 
which gradually increases in size and apparently contains a bubble 
of gas. The upper portion of the culture-medium at the same time 
becomes liquefied, while the lower portion remains solid for days. 
In a suspended drop spirochsetae-like spirals are observed at the 
margins, which often present as many as twenty distinct arches. 1 

1 R. Koch, Berlin, klin. Woch., 1884, vol. xxi. pp. 477, 493, 509. . 



PLATE XV. 



FIG. 



Ufa® J ^ 



#7 



Spirillum of Asiatic Cholera. Impression Cover-slip from a Colony 
Thirty-four Hours Old. (Abbott.) 



FIG. 2. 




Bacillus of Finkler and Prior. (Cornil and Babes.) 



FIG. 3 






Bacillus of Typhoid Fever from a Culture Twenty-four Hours Old, 
on Agar-agar. (Abbott.) 



PATHOLOGY OF THE FECES. 325 

Closely related to Koch's comma-bacillus is the bacillus of FinJder 
and Prior, discovered in 1884 and 1885 (Plate XV., Fig. 2). 
This is, however, readily distinguished from the former by the 
following characteristics : it is larger and thicker than the comma- 
bacillus ; the colonies on gelatin plate-cultures show equally round 
and sharp-edged forms, which present a granular appearance under 
a low or medium power, and are usually of *a brown color. The 
organism liquefies gelatin very rapidly, a penetrating, excessively 
fetid odor being developed at the same time. In stab-cultures 
the bacillus of cholera Asiatica forms a funnel-shaped depression, 
while the bacillus of Finkler and Prior forms a stocking-like 
depression. 1 

In this connection the green bacillus of Le Sage, discovered in 
certain forms of infantile diarrhoea, must briefly be referred to, the 
stools, as has been mentioned, being of a grass-green color. The 
production of this pigment in cultures is one of the characteristics of 
the organism ; when injected into the intestines of animals it is said 
to produce diarrhoea and a catarrhal inflammation of the mucous 
membrane. 

Booker 2 has described nine different bacilli as occurring in cases of 
infantile diarrhoea. Seven of these closely resemble the Bacillus coli 
communis. Bacillus "A" is a bacillus with rounded ends, measur- 
ing from 3 fi to 4 ii in length by 0.7 jul in breadth. It is motile and 
liquefying. Colonies on agar and potato present a dirty-brown 
color. 

The typhoid bacillus, discovered by Eberth 3 in 1880 in the ab- 
dominal organs of patients dead with typhoid fever, is unfortunately 
not so readily recognized in the feces as the organisms just described. 
This is owing to the intimate relation which apparently exists be- 
tween the bacillus in question and the Bacillus coli communis, with 
which it has many properties in common. A few years ago Eisner 
suggested a method which, it was hoped, would effectually overcome 
this difficulty, and in the hands of numerous observers good results 
were obtained. Widal's agglutination test, however, which was 
almost simultaneously introduced, diverted attention from the study 
of the feces, and Eisner's work has practically been forgotten. 

In the meantime Widal's test has been carefully investigated, 
and although the reaction must unquestionably be considered as a 
specific reaction of typhoid fever, its value in diagnosis is neverthe- 

1 Finkler, Deutsch. med. Woch., Tageblatt der Naturforscherversamrnlung, 1884, 
vol. x. p. 36, and 1885, p. 438. Finkler u. Prior, Erganzungshefte z. Centralbl. f. allg. 
Gesundheitspflege, 1885, vol. i. 

2 W. D. Booker, " A Bacteriological and Anatomical Study of the Summer Diarrhoeas 
of Infants," Johns Hopkins Hosp. Rep., vol. vi. 

3 Eberth, Virchow's Archiv, 1881, vol. lxxxiii. p. 486. 



326 THE FECES. 

less limited (see page 100). As a consequence, further attempts 
have been made to discover a method which will enable the general 
practitioner to establish definitely the diagnosis of typhoid fever at 
an early stage of the disease. Whether or not Eisner's method 
(v. i.) has been deservedly abandoned, further investigations will 
show. At the present time another procedure, which was suggested 
by Piorkowski, is attracting widespread attention, as it is claimed 
that with this method the diagnosis can be made within twenty -four 
hours. 

Piorkowski's Method. 1 — The necessary culture-medium is pre- 
pared as follows : normal urine of a specific gravity of about 1.020 
is allowed to stand until the reaction has become alkaline ; it is then 
treated with 0.5 per cent, of peptone and 3.3 per cent, of gelatin, 
boiled for one hour, and filtered immediately into test-tubes with- 
out any further application of heat. The test-tubes are closed with 
cotton, sterilized for fifteen minutes in a steam sterilizer at 100° C, 
and resterilized after twenty-four hours for ten minutes. 

To examine the feces, one tube is inoculated with 2 oesen of the 
fecal matter, which should be as fresh as possible. From this tube 
4 oesen are transferred to a second tube, and a third is inoculated 
with from 6 to 8 ceesen from the one preceding. Plates are finally 
prepared and kept at a temperature of 22° C, as the presence of 
so small an amount of gelatin does not permit of exposure to higher 
temperatures. After sixteen to twenty -four hours an examination is 
made with a low power. At the expiration of this time the colonies 
of the colon bacillus appear as round, yellowish-brown, and finely 
granular specks, with well-defined borders, while the typhoid colo- 
nies show a peculiar flagellate appearance, from two to four fine 
colorless radicles usually starting from a light, highly refractive 
central focus. After forty-eight hours the radicles have greatly 
extended, and after forty-eight to fifty-six hours the colonies are 
perfectly developed and present a picture which strongly suggests the 
appearance of radishes, minute interweaving branches being given 
off in every direction, while no difference can be observed at this 
time between typhoid and colon bacilli which have been grown for 
control in 10 per cent, normal or bouillon-gelatin. 

Piorkowski claims that he has thus been able to demonstrate the 
presence of typhoid bacilli in infected drinking-water, and in the 
feces of typhoid fever patients at a time when a positive result could 
not yet be obtained with WidaPs test. Recent reports bear out the 
claims of Piorkowski, and the method can hence be recommended in 
doubtful cases. 2 

1 Piorkowski, " Ein einfaches Verfahren z. Sicherstellung d. Typkusdiagnose," 
Berlin, klin. Wock., 1899, p. 145. 

2 A. Sckiitze, "Ueber d. Nackweis.v. Typkusbacillen in den Faeces," Zeit. f. klin. 
Med., vol. xxxviii. p. 39. 



PATHOLOGY OF THE FECES. 327 

Elsner's Method. 1 — The culture-medium is prepared as follows : 
an aqueous extract of potato (500 grammes to the liter) is treated 
with 10 per cent, of gelatin and boiled. The solution is then treated 
with 2.4 to 3.2 c.c. of a one-tenth normal solution of sodium 
hydrate, in order to secure the necessary degree of acidity, and then 
filtered and sterilized. 

When needed, a portion is placed in an Erlenmeyer flask and 
treated with 1 per cent, of potassium iodide. The mixture is inocu- 
lated with fecal material and the necessary plates prepared. Upon 
this medium only a few species of bacteria will grow, principally the 
Bacillus coli and the typhoid bacillus. After twenty-four hours the 
Bacillus coli colonies are already mature, while the typhoid colonies 
can scarcely be made out with a low power. After forty-eight hours, 
however, they appear as small, highly refractive, extremely fine, 
granular colonies, closely resembling drops of water, which can be 
readily distinguished from the large, much more granular, brownish 
colonies of the Bacterium coli. This difference is brought out par- 
ticularly well if diluted plates have been prepared. 

Brieger, 2 who carefully repeated the experiments of Eisner, states 
that typhoid bacilli are found in abundance in the stools so long 
as fever exists, but with approaching convalescence they diminish in 
number and ultimately disappear. If, notwithstanding the absence 
of fever, bacilli are found in notable numbers during convalescence, 
a relapse may be anticipated. 

In pure cultures the typhoid bacilli present the following features : 
they occur in the form of rods of almost one-third the size of a red 
blood-corpuscle, or in threads composed of several rods joined end 
to end (Plate XV., Fig. 3). Their ends are rounded ; their length 
is equivalent to about three times their breadth. They are actively 
motile and provided with polar as well as lateral flagella. They 
grow very readily on bouillon-peptone gelatin, and after twenty-four 
hours colonies begin to appear. When slightly magnified, these 
present a faintly yellowish color ; macroscopically they are barely 
visible. When kept at a temperature of 37° C. the formation of 
spores may be observed, especially when the organism is grown on 
media colored with phloxin-red or benzopurpurin. Gelatin is not 
liquefied ; the growth is white and delicate, both along the line of 
the stab and on the surface. Cultivation in glucose-bouillon, or 
glucose-agar, does not give rise to the formation of gas, but after 
twenty-four hours the entire fluid becomes turbid. Milk is ren- 
dered feebly acid, but is not coagulated. No indol reaction is ob- 
tained when the organism is grown on peptone-containing media. 
On potato a very faint, whitish, almost invisible growth takes place. 
When grown on gelatin or agar that has been colored with neutral 

1 Eisner, Zeit. f. Hyg. u. Infektionskrank., 1H95, vol. xxi. p. 25. 

2 Brieger, Deutsch. med. Woch., 1895, vol. xxi. p. 835. 



328 THE FECES. 

red, the typhoid bacillus causes no change in color. Absolute 
identification is possible by means of Pfeiffer's agglutination-test 
(see WidaFs reaction). 

In cases of paratyphoid infection the corresponding organism may 
be found in the feces (see Blood). 

Tubercle bacilli, when present in the feces, are indicative of intestinal 
tuberculosis, providing they are observed upon repeated examination 
and there are clinical symptoms pointing to the bowels as the seat 
of the disease ; otherwise they may be referable to swallowed sputa. 
They may be demonstrated as described in the chapter on Sputum. 

The Bacillus coli communis, 1 while constantly present in normal 
feces, is described at this place, as modern investigations have shown 
that it may at times develop pathogenic properties. It has been 
found in the pus in cases of purulent perforating peritonitis, angio- 
cholitis, pyelonephritis, etc., and, as indicated elsewhere, at times 
forms the nucleus of gall-stones. It occurs in the form of delicate 
or coarse rods, measuring about 0.4 /i in length, which manifest a 
certain degree of motility, due to the presence of one or two polar 
flagella. The organism is stained by the usual anilin dyes, and is 
decolorized by Gram's method. The colonies upon gelatin closely 
resemble those of the bacillus of typhoid fever, forming small whitish 
specks in the gelatin, and delicate films with serrated borders upon 
the same medium, which, moreover, is not liquefied. On potato the 
organism forms a brownish pellicle, while the growth of the typhoid 
bacillus is nearly transparent. As in the case of the cholera bacillus, 
the nitrosoindol reaction can be obtained when the organism is 
grown upon peptone-containing media. In solutions of glucose 
active fermentation takes place. Litmus milk is rendered acid and 
is coagulated. Important also is the behavior of the organism 
when grown on gelatin or agar that has been colored with neutral 
red ; in contradistinction to the typhoid bacillus, the colon bacillus 
then causes an intense green fluorescence. 

The Bacterium lactis aerogenes (Escherich) closely resembles the 
organism just described, and may also at times develop pathogenic 
properties. It was recently found in a case of pneumaturia and in 
one of idiopathic bacteriuria. It is seen quite constantly in the 
stools of sucklings, but may also be met with in those of adults. 
It occurs in the form of rather stout rods, which frequently lie in 
pairs, resembling diplococci. The organism is non-motile. Like 
the Bacillus coli communis, it is decolorized by Gram ? s method. In 
plate-cultures it forms a dense white film ; in stab-cultures a chain 
of white colonies resembling beads is seen. In the latter, moreover, 
if the stab is closed, bubbles of gas will be seen to form, which 
rapidly increase in number and size. Milk is coagulated in large 
lumps in twenty-four hours ; at the same time, the formation of 

1 Flugge, Die Microrganismen. 



PATHOLOGY OF THE FECES. 329 

gas is much more intense than in the case of the Bacillus coli 
communis. 

The Bacillus pyocyaneus has within recent years been isolated from 
the stools of dysenteric patients, and has been proved the cause of 
several epidemics. The organism in question is a small motile bacil- 
lus measuring from 1 // to 2 fi in length by 0.3 fi to 0.5 fi in breadth. 
It sometimes occurs in short chains, but is usually single. It is 
stained with the common anilin dyes, and is decolorized with Gram's 
method. It grows on the usual culture-media, and liquefies gelatin. 
In 2 per cent, glucose-bouillon no fermentation takes place. Litmus- 
milk is curdled in about forty-eight hours. Some varieties produce 
indol. Most characteristic is the production of certain pigments, 
viz., pyocyanin and a fluorescent bluish-green pigment which is 
common to almost all varieties. 1 

Bacillus acidophilus, Moro. 2 This organism has recently been 
described by Moro as occurring in the stools of breast-fed infants, in 
which it normally prevails over all other forms ; under pathological 
conditions, on the other hand, as also in the stools of children, 
which have been fed with cows' milk their number is found dimin- 
ished, while the members of the coli-group enter into the foreground. 
Beyond the stools, the bacillus has been found in the outer portion 
of the secretory duct of the human mammary gland, in the milk, 
and the skin of the nipple and its immediate surroundings. It is 
apparently not pathogenic. 

The organism occurs in the form of slight rods measuring 1.5 p. 
to 2 f± in length, by 0.6 /i to 0.9 p. in breadth. It is non-motile. 
It is not decolorized by Gram's method, but loses this property after 
from thirty-six hours to nine days. The best growths are obtained 
on beer wort bouillon and common bouillon when acidified with a 
mineral acid ; the acidity of 10 c. c. of the medium may correspond 
to 10 c.c. of a decinormal solution of potassium hydrate. The 
optimum temperature is 37° C.; between 20° C. and 22° C. no 
growth occurs. On the various agar-slants imperfect development 
takes place ; on potato the organism does not grow. It is an active 
acid-producer, but does not give rise to the formation of gas ; with 
Escherich's stain it is colored blue. 

Escherich's Stain. — This stain is now extensively used by podiat- 
rists in order to ascertain any deviations from the normal in the flora 
of the feces. Under strictly normal conditions the bacilli which are 
found in the stools of breast-fed children are thus nearly all colored 
blue (see above), while red bacilli are but little numerous. In the 
case of infants, on the other hand, which are fed exclusively on cows' 

1 A. J. Lartigau, " A Contribution to the Study of the Pathogenesis of the Bacillus 
Pyocyaneus," etc., Jour. Exper. Med., 1898, No. 6. 

2 Moro, " Ein Beitrag zur Kenntniss der normalen Darmbacterien des Sanglings," 
Jahrbuch f. Kinderheilk., vol. lii. Also: "Ueber die nach Gram farbbaren Bacillen 
d. Sauglingstuhles," Wien. klin. Wocb., 1900, No. 5. 



330 THE FECES. 

milk the red bacilli predominate, while in mixed feeding the blue 
enter into the foreground in about the proportion in which breast- 
milk is employed. The red bacilli belong to the coli-group. These 
further predominate, or may be found exclusively, if for any reason 
intestinal digestion is impaired. Staphylococci, streptococci, etc., 
when simultaneously present, are in either event stained blue. In 
staphylococcus enteritis the blue bacilli which normally exist in the 
stools of breast-fed infants are almost entirely replaced by staphylo- 
cocci. At the beginning of the enteritis they are not numerous, but 
they increase during the progress of the disease, and finally disappear 
when the child recovers. 

In staining, the following solutions are employed : 

1. An aqueous solution of gentian-violet (5 : 200). This is boiled 
for one-half hour and is then filtered ; it keeps for a long time. 

2. A mixture containing 11 parts of absolute alcohol and 3 parts 
of oil of anilin. 

(1) and (2) are mixed in the proportion of 8.5 : 1.5 ; the resulting 
solution keeps for from two to three weeks, but not longer. 

3. A solution of iodo-potassic iodide containing 1 part of iodine 
and 2 parts of potassium iodide in 60 parts of water. 

4. A mixture of equal parts of oil of anilin and xylol. 

5. A concentrated alcoholic solution of fuchsin, diluted with an 
equal volume of absolute alcohol. 

A bit of the stool is spread upon a slide in as thin a layer, as 
possible. After drying in the air the specimen is fixed by passing 
through the flame of a Bunsen burner. It is then stained for a few 
seconds with the mixture of (1) and (2), blotted, placed in the 
iodine solution for a few seconds, blotted again, decolorized with (4) 
until a notable extraction of color no longer occurs. It is washed 
with xylol, dried, and finally stained for a few seconds with the 
fuchsin solution, washed with water, blotted, and is then ready for 
examination. 1 

Proteus vulgaris, Hauser. This organism, while usually regarded 
as non-pathogenic, should be numbered among the bacteria which 
may at times develop pathogenic properties. Baginsky and Booker 
have frequently found it in the stools in cases of infantile summer 
diarrhoea. Escherich observed it at times in the meconium. 
Brudzinski examined the dyspeptic and fetid stools of a number 
of artificially fed infants in Escherich 7 s clinic, and in all the cases 
found the proteus. Others have encountered it in inflammatory 
conditions of exposed surfaces, in appendicitis, in perforative peri- 
tonitis, and even in closed abscesses, either alone or in association 
with other bacteria (Welch). A mixed infection with the proteus 
and Loffler's bacillus has also been observed. The organism forms 
rods, measuring about 0.25 ju in diameter, while their length is 

1 Moro, loc. cit. 



PATHOLOGY OF THE FECES. 331 

variable ; at times a more roundish form is observed ; at others 
rods measuring from 1.25 // to 3.75 fx in length, or even long 
threads. They are readily stained, but are easily decolorized by 
alcohol or Gram's method. Most characteristic is their growth upon 
nutrient gelatin. At the temperature of the room little depressions will 
be observed after six to eight hours, which are surrounded by a narrow 
zone of bacilli from which a thin, wide film, provided with irregular 
projections, extends over the culture-medium. From this film 
islets become separated, which slowly extend over the gelatin and 
cause its liquefaction. The organism is motile. It decomposes 
urea and causes albuminous putrefaction. The nitroso-indol reaction 
is readily obtained in bouillon-cultures. 1 In boiled milk the organ- 
ism grows well, while in fresh milk it develops only irregularly, and 
in acid milk no growth takes place at all. 

Bacillus dysenteriae, Shiga. This organism is now generally re- 
garded as the specific cause of the common form of acute dysentery 
which prevails not only in the tropics, but also in the United States 
and Europe. It was discovered by Shiga in Japan, in 1897, and 
is identical with the organism obtained by Flexner and Strong in the 
Philippines and Porto Rico, by Vedder and Duval in the United 
States, and by Kruse in Germany. From the researches of Bassett 
and Duval it further appears that the same bacillus is also responsi- 
ble for the common form of infantile summer diarrhoea which pre- 
vails in warm countries. 

The bacillus in question is a short rod with rounded ends, which 
much resembles the typhoid bacillus and most members of the colon 
group. It is probably non-motile so far as active locomotion is 
concerned, but it is possessed of a high degree of molecular motion. 
It stains with the usual basic dyes and is decolorized by Gram's 
method. 

Upon gelatin plates at room temperature there appear, after a few 
days, small round dots, which, magnified under low powers, are 
slightly yellow and finely granular. After a few days they increase 
in size ; the middle portion of the colonies then appears darker under 
a low power, while the outer zone appears brighter and more seed-like. 
The superficial and deeper colonies show no marked variation. In 
stab-cultures of gelatin a whitish strand forms the whole length of 
the stab. The gelatin is not liquefied. 

After twenty-four hours in the incubator single colonies upon 
slanted agar appear moist, bluish, and partially translucent. After 
two days they present a combination of a middle dark and a periph- 
eral bright, sharply defined zone. 

The growth on glycerin-agar is slightly more abundant than on 
ordinary agar. The organism grows on blood-serum without lique- 
fying it. 

i Fliigge, loc. cit. 



332 THE FECES. 

In the stab-cultures on glucose-agar there is farmed along the 
whole line of the puncture a thick gray-white strand without the 
development of gas. Upon potato after twenty -four hours in the 
incubator there is hardly any perceptible growth, only the surface 
appears slightly shiny. After two days this changes to a yellow 
brown. In the course of a week the growth is heavier and of a 
deeper brown color. Bouillon cultures show after a day in the 
incubator a somewhat intense cloudiness, with a moderate precipitate. 
No pellicle is formed on the surface. No indol reaction is present. 
Litmus-milk after twenty -four hours appears reddish ; otherwise, 
however, it undergoes no change. The milk never coagulates. 

The bacillus is pathogenic for mice, rabbits, and guinea-pigs. 
It is agglutinated by the patient's blood-serum, and it is interesting 
to note that this reaction is obtained only with cases definitely known 
to have been infected with the micro-organism in question. 

Isolation of Shiga's Bacillus from the Feces. — The fecal matter is 
collected on a sterile pad, or, still better, obtained from the rectum 
by curettage. A bouillon culture is prepared and from this agar 
tubes are inoculated as soon as possible. The agar should be just 
acid to phenolphthalein (slightly alkaline to litmus), and is plated 
at once. Ten plates, variously diluted, are conveniently used. After 
twenty-four hours in the incubator at 37°-38° C. all colonies are 
marked on the plates which have developed by that time. The 
plates are returned to the incubator. After further twenty-four 
hours tubes of glucose agar and litmus-mannite agar are inocu- 
lated from those colonies which have grown in the second twenty- 
four hours — i. e. 9 those colonies which have not been marked. At 
the end of another twenty-four hours in the incubator all those tubes 
are rejected in which fermentation has taken place. From those 
tubes in which this has not occurred, litmus milk, litmus mannite, 
and bouillon are inoculated. The Shiga bacillus will at first render 
the milk slightly acid, but later it becomes alkaline. Litmus mannite 
remains unchanged with the Shiga strain, while the Harris type (the 
American form) turns it red. Ultimate identification is made by the 
agglutination test in various dilutions (1 : 50 to 1 : 100) reading the 
results after two hours. 

Literature. — K. Shiga, Centralbl. f. Bakt., Parasit. u. Infectionskrankh., 1898, 
vol. xxiv. E. P. Strong and Musgrave, "Preliminary Note regarding the JEtiology 
of the Dysenteries of Manila." Eeport of the Surgeon-General of the Army, Washing- 
ton, 1900, p. 251. S. Flexner, "On the Etiology of Tropical Dysentery," Bull. Johns 
Hopkins Hosp., ]900, p. 231. Vedder and Duval, " The Etiology of Acute Dysentery 
in the United States," Jour. Exper. Med., vol. vi. p. 181. Duval and Bassett, Am. Med., 
1902, iv. p. 417 (preliminary report). 






CHEMISTRY OF THE FECES. 333 

CHEMISTRY OF THE FECES. 

Mucin. — According to Hoppe-Seyler, mucin is a constant con- 
stituent of the feces, both under physiological and pathological 
conditions. Normally, however, it is never possible to recognize its 
presence either with the naked eye or with the microscope. In 
order to demonstrate the presence of mucin the feces are digested 
with water and treated with an equal volume of milk of lime; 
the mixture is allowed to stand for several hours, when it is filtered 
and the filtrate tested Avith acetic acid. In the presence of mucin 
a cloud develops upon addition of the acid. 

Albumin is demonstrated in the feces by treating repeatedly 
with water slightly acidified with acetic acid. The filtrate is then 
examined for albumin according to methods given elsewhere (see 
Urine). Under normal conditions these reactions prove negative. 
Pathologically, serum-albumin has been observed in cases of typhoid 
fever and chlorosis. 

Peptones (albumoses) are normally absent from the feces. They 
have been observed in typhoid fever, dysentery, tubercular ulcera- 
tion, purulent peritonitis with perforation into the gut, atrophic 
cirrhosis, and carcinoma of the liver. Acholic stools are also usually 
rich in peptones. 

The peptones are demonstrated in the following manner : the 
feces are digested with water, so as to form a thin mush ; they are 
then boiled, filtered while hot, and the filtrate examined for albumin, 
so as to be sure that all of this has been removed. The mucin is 
removed by treating with lead acetate, when the filtrate is examined 
for peptones as described in the chapter on Urine (which see). 

Carbohydrates. — Of the carbohydrates, starch, glucose, and 
certain gums may be found. In order to demonstrate these the feces 
are boiled w T ith water, filtered, and evaporated to a small volume. 
This solution may now be tested with phenylhydrazin or Troramer's 
reagent for glucose (see Urine), and with a solution of iodo-potassic 
iodide for starch (see Saliva, page 199). The residue is extracted 
with alcohol and ether, as described under the heading of fatty acids, 
and then with water. The filtrate of the aqueous extract is con- 
centrated, boiled with dilute sulphuric acid, and then over-saturated 
with sodium hydrate. This mixture is treated with cupric sulphate 
and boiled, in order to test for dextrin and gums. 

In normal breast-fed infants sugar is only demonstrable in traces 
in the stools. Langstein l finds that the presence of more than traces 
of glucose in the stools of milk-fed infants may be regarded as a 
diagnostic symptom of the localization of a catarrhal process in the 
duodenum. 

Bile -pigment, which is normally absent from the feces, occurs in 
1 L. Langstein, Jahresb. f. Kinderheilk., vol. vi. Heft 3. 



334 THE FECES. 

large amounts in catarrhal conditions of the small intestine, and may 
be demonstrated by Gmelin's method, viz., a drop of the filtered 
liquid, or a particle of highly colored fecal matter, is brought into 
contact with a drop of fuming nitric acid, when the yellow color will 
be seen to pass through the various shades of the spectrum, the 
green shade being the most characteristic. At times, however, it is 
not possible to obtain a positive reaction in this manner, although 
bile-pigment is present. In such cases the examination should be 
conducted under the microscope, and attention directed to bile-stained 
epithelial cells, leucocytes, particles of mucus, and crystals. 

Whenever there is increased intestinal putrefaction the fatty acids, 
phenol, indol, and skatol will, of course, be found in increased 
amounts. 1 

Ptomains. — Of ptomains, only two have been isolated from the 
feces, under pathological conditions, viz., putrescin and cadaverin. 
They have been found in Asiatic cholera, in cholerina, dysentery, 
and in connection with cystinuria. In cholera and cystinuria their 
amount may be quite large. Baumann and v. Udranszky thus 
obtained 0.5 gramme of the benzoylated compounds from the col- 
lected feces of twenty-four hours. In cholera the cadaverin seems 
to predominate, while in cystinuria more putrescin is found. 1 

To isolate the diamins in question, the feces are digested with 
alcohol which has been acidified with sulphuric acid. The alcoholic 
extract is evaporated, the residue dissolved in water, and further 
benzoylated, as described in the section on Urine. 

MECONIUM. 

By meconium are meant those masses which are first excreted from 
the bowel after birth. It is a thick, tenacious, greenish-brown mate- 
rial, ivhich has accumulated during the intra-uterine life of the 
infant. Microscopically, a few cylindrical epithelial cells, a few 
fat-droplets, numerous cholesterin-crystals, bilirubin-crystals, and 
lanugo-hairs are found. 

Micro-organisms are absent, but soon after suckling has com- 
menced they appear in abundance. The most important of those 
which are then constantly present are the Bacillus lactis aerogenes, 
which predominates in the small intestine, and the Bacillus coli 
communis, which is found more particularly in the large intestine. 
Both have already been described (see page 328). 2 

In addition to these, the Proteus vulgaris, Streptococcus coli brevis, 
Micrococcus ovalis, tetragencoccus, Torula cerevisise, Torula rubra, 
and a few less important micro-organisms have been found. 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., Phila., 1901. 

2 A. E. Austin, "The Chemical Examination of the Feces for Clinical Purposes," 
Phila. Med. Jour., 1900, j). 551. 



MECONIUM. 335 

Chemically, meconium contains bilirubin in considerable amount 
(recognizable by Gmelin's reaction), biliary acids, fatty acids, chlo- 
rides, sulphates, phosphates of the alkalies and their earths. It 
does not contain urobilin, glycogen, peptones, lactic acid, tyrosin, 
or leucin. 

An idea may be formed of its composition from the following 
analysis of Zweifel i 1 

Water' 79.8-80.5 per cent 

Solids 19.5-20.2 " 

Mineral matter 0.978 " 

Cholesterin 0.797 

Fats 0.772 

1 Hellstrom, Arch. f. Gynak., 1901, vol. lxiii. Heft 3. 



CHAPTEE V. 

THE NASAL SECRETION. 

In the nasal secretion, which normally is small in amount, trans- 
parent, colorless, odorless, tenacious, and of a slightly saline taste, 
pavement-epithelial cells in large numbers, ciliated epithelial cells, as 
well as some leucocytes and an enormous number of micro-organisms, 
are found (Fig. 77). Its reaction is alkaline. 

Fig. 77. 




Epithelial cells and mucous corpuscles found in the nasal secretion. 

In acute coryza the amount is diminished at first, but soon a very 
copious secretion occurs, which contains numerous epithelial cells 
and micro-organisms. When complicated with an ulcerative condi- 
tion pus is observed in considerable amount. 

Occasionally, as in cases of traumatism, cerebral tumors, etc., 
cerebrospinal fluid is discharged through the nose, and may be 
recognized by the fact that it is free from albumin and contains a 
substance which reduces Fehling's solution. 

Of pathogenic organisms, the tubercle bacillus and the bacillus of 
glanders may occur in ulcerative diseases of the nose, their presence 
indicating the existence of the corresponding affection. In ozsena a 
large diplococcus has been described by Lowenberg, which is said to 
be characteristic of the disease. Oidium albicans has been observed 
in rare cases. The Meningococcus intracellularis of Weichselbaum, 
which is now quite generally regarded as the cause of epidemic 
cerebrospinal meningitis, has also been demonstrated in the nasal 
secretion of healthy individuals. This fact helps to explain the 
origin of those cases of meningitis which develop after injuries to 
the skull. 

336 



THE NASAL SECRETION. 337 

Ascarides and other entozoa have also been found. Charcot- 
Leyden crystals (see page 364) have been observed in the nasal 
secretion in cases of bronchial asthma and in connection with 
nasal polypi. Their presence is usually accompanied by the simul- 
taneous occurrence of eosinophilic leucocytes. 

Literature. — Eeimann, Baumgarten's Jahresber., 1888, vol. iii. p. 417. Lowenberg, 
Deutsch. med. Woch., 1885, vol. xi. p. 6, and 1886, vol. xii. p. 446. Tost, Ibid., p. 161. 
Gerber u. Podack, Deutsch. Arch. f. klin. Med., 1895, vol. liv. p. 262. Leyden, Deutsch. 
med. Woch., 1891, vol.xvii. p. 1085. Sticker, Zeit. f. klin. Med., 1888, vol.xiv. p. 81. 
Nothnagel, Wien. med. Blatter, 1888, Nos. 6, 7, 8. 

22 



CHAPTER VI. 
THE SPUTUM. 

GENERAL TECHNIQUE. 

The sputum should be collected in receptacles so constructed as 
to permit of their complete and easy disinfection. The paper spit- 
cups (Fig. 78) which have been introduced within late years are 
admirably adapted to this purpose, as they may be destroyed imme- 
diately after use. 

Fig. 78. 





Sanitary spit-cups. 

When working ivith sputa which are known or suspected to be of 
tubercular origin, the greatest care should be exercised to keep the expec- 
toration from drying and becoming disseminated in the air. Negligence 
in this respect may result in the most serious consequences. 

The macroscopical examination of sputa is most conveniently 
carried out by placing small portions of the material upon a plate of 
ordinary window-glass, of suitable size, which has been painted black 
upon its lower surface, and covering the same with a second, smaller 
plate. If it is desired to examine individual constituents which 
have been discovered in this manner, the upper plate is slid off until 
the particle in question is uncovered, when it may be removed to a 
microscopical slide and examined under a higher power. 

It is also very convenient to have a portion of the laboratory 
table painted black, when unstained plates of glass may be utilized. 
If these measure about 15 by 15 cm. and 10 by 10 cm., respectively, 
fairly large quantities of sputum may be examined in situ with a 
low power. 

338 



GENERAL CHARACTERISTICS OF THE SPUTA. 339 

GENERAL CHARACTERISTICS OF SPUTA. 

Amount. — The amount of sputum expectorated in the twenty- 
four hours varies within wide limits, depending largely upon the 
nature of the disease. Thus, only a few cubic centimeters may 
be eliminated, or the amount may reach 600 to 1000 c.c, and even 
more. Very large quantities are expectorated in cases of pulmonary 
hemorrhage and oedema of the lungs, sometimes following thoracen- 
tesis, also following perforation of accumulations of pus from the 
thoracic or abdominal cavities into the respiratory passages ; further- 
more, in cases in which large vomicae of tubercular or gangrenous 
origin exist, and finally in cases of abscess of the lung, bronchiectasis, 
and even in simple bronchial blennorrhoea. In incipient phthisis, 
acute bronchitis, and in the first and second stages of pneumonia, c^ 
the other hand, the amount is usually small. 

In private practice, as well as in hospital work, an idea should 
always be formed of the amount expectorated in the twenty-four 
hours, especially in cases in which this is abundant. It is apparent 
that a copious and long-continued expectoration cannot continue 
without exerting very detrimental effects upon the patient's general 
nutrition ; in cases of pulmonary phthisis, for example, Renk has 
shown that 3.8 per cent, of all nitrogen eliminated in such cases is 
removed in this manner. Lenz in his recent experiments found even 
5 per cent. 

Consistence. — The consistence of the sputum corresponds, in a 
general way at least, to its amount, and may vary from a liquid to 
a highly tenacious state. The cause of the tenacity of the sputum 
is but imperfectly understood. The mucin present does not appear 
to be the most important factor, as it has been observed to occur 
in diminished amount in pneumonic sputa, which are noted for their 
high degree of tenacity. Kossel l has suggested that the phenomenon 
may be due to the presence of nucleins or nuclein derivatives, while 
others again refer it to the presence of abnormal albuminous 
bodies of unknown character. However this may be, sputa are not 
infrequently seen where it is possible to invert the cup without 
losing a drop of its contents. This is observed especially in cases 
of acute croupous pneumonia up to the time of the crisis, pro- 
viding that a catarrh of the bronchi does not exist at the same 
time. It is noted, furthermore, immediately after an attack of acute 
bronchial asthma, and also in the initial stage of acute bronchitis. 
In cases of oedema of the lungs, on the other hand, the sputa are 
liquid and present the general characteristics of blood-serum, being 
covered, like all albuminous liquids when brought into contact with 
the air, by a frothy surface-layer. The sputa observed in cases of 
acute pulmonary gangrene, pulmonary abscess, putrid bronchitis, and 
1 Kossel, Zeit. f. klin. Med., 1888, vol. xiii. p. 152. 



340 THE SPUTUM. 

following perforation into the lungs of an empyema or an accumula- 
tion of pus situated beneath the diaphragm, are fluid and consist of 
pure pus. 

Color. — The color of the sputa may vary greatly. They may be 
perfectly clear and transparent, gray, yellow, green, red, brown, and 
even black. Purely mucoid expectoration is almost transparent and 
colorless, as is also the sputum of pulmonary oedema when not mixed 
with blood or pus. 

The larger the number of leucocytes the more opaque does the 
sputum become, assuming at first a white, then a yellow, and finally 
a greenish color, the two latter colors being usually indicative of the 
presence of pus. Green sputa, however, may also be observed when 
bile-pigment has become admixed with the sputa, as in cases of perfo- 
ration of a liver-abscess into the lung. Green sputa may also be 
observed in cases of jaundice, and especially in pneumonia when 
accompanied by icterus. In cases of amoebic liver-abscess with 
perforation into the lung the sputa present a color resembling 
anchovy sauce, which is very characteristic. In one case I recog- 
nized the nature of the disease by simple inspection of the sputa. 1 

The inhalation of particles of carbon gives the sputum a grayish 
or even a black color ; the same or an ochre-yellow or red color is 
observed in cases of siderosis. 

A red color is usually indicative of the presence of blood, the in- 
tensity of the shade depending upon the character of the disease. 
It is seen especially after the formation of cavities, in caseous pneu- 
monia, in incipient phthisis, heart-disease, etc. In general, it may 
be said that a clear, bright-red color indicates an arterial, a dark- 
red or bluish-red a venous origin of the hemorrhage. The exact 
shade will depend upon the length of time that the blood, no matter 
what its origin may be, has remained in the lungs. In pulmonary 
gangrene a dirty brownish-red color is observed, owing to the pres- 
ence of methsemoglobin, and, to some extent also, of hsematin. 
Quite characteristic is a chocolate color, which is observed when a 
croupous pneumonia terminates in necrosis and gangrene. Equally 
characteristic is the rusty and prune-colored expectoration seen in 
cases of pneumonia. Occasionally a breadcrust-brown color is 
observed in cases of gangrene and abscess of the lung, which is 
quite characteristic, the color being due to the presence of hseniatoidin 
or bilirubin. 

Rust-colored punctate or striped sputa, moreover, are said to be 
diagnostic of brown induration of the lung. 

Odor. — Most sputa are odorless. Under certain conditions, how- 
ever, there may be a very marked odor. In cases of pulmonary 
gangrene or putrid bronchitis the odor is of a kind never to be for- 
gotten, the stench, indeed, being frightful. A somewhat similar, 

1 See Johns Hopkins Hosp. Bull., November, 1890, 



GENERAL CHARACTERISTICS OF SPUTA. 341 

slightly sweetish odor is observed in certain cases in which putre- 
factive organisms have entered the lungs, and there exert their action 
upon the accumulated sputa, in the absence of gangrene, as in cases 
of bronchiectasis, perforating empyema, and where ulcerative proc- 
esses are taking place in the lungs, whether these be of tubercular 
origin or not. An odor like that of old cheese is occasionally 
observed in cases of perforating empyema ; under such conditions 
tyrosin is usually found. This body, however, has nothing to do 
with the odor of the sputa ; both factors are merely indicative of 
certain putrefactive changes going on in the lungs. According to 
Leyden, the occurrence of tyrosin in sputa is usually indicative of 
the perforation of an old accumulation of pus into the lungs. 

Specific Gravity. — The specific gravity of sputa varies within 
wide limits ; mucous sputa have a specific gravity of 1.004 to 1.008, 
purulent sputa one of 1.015 to 1.026, and serous sputa one of 1.037 
or more. 

Configuration of Sputa. — As a general rule, the following forms 
of sputa, which may be termed pure sputa, present a homogeneous 
appearance : 

Mucoid sputa, 

LZ^C' \ Homogeneous sputa, 

Sanguineous sputa, J 

with one exception, perhaps — the typically rusty sputa of croupous 
pneumonia ; while mixtures of any two or three of these may be 
classed as heterogeneous sputa : 

Mucopurulent sputa, "1 

Mucoserous sputa, [ Heterogeneous sputa. 

Serosangumeous sputa, & r 

Sanguino-mucopurulent sputa, J 

The so-called sputum crudum of the first stage of acute bronchitis 
may be regarded as an example of a purely mucoid sputum. A 
purely purulent sputum is usually indicative of the perforation of an 
empyema or any other accumulation of pus into the lungs or bronchi, 
of pulmonary abscess, or of bronchial blennorrhea. A purely serous 
sputum is found in cases of pulmonary oedema, and a purely hemor- 
rhagic sputum in cases of severe pulmonary hemorrhage. 

Of the heterogeneous sputa, the most important are the so-called 
nummular sputa of the second and third stages of phthisis. These 
are characterized by the fact that when thrown or expectorated into 
water they sink to the bottom, and there form coin-like disks, from 
which property they have received their name. Such sputa are 
mucopurulent in character, and contain a focus of almost pure pus 
imbedded in a more or less homogeneous mass of mucus. Quite 
different from these are the so-called sputa globosa of the ancients, 
which consist of fairly dense, roundish, grayish-white masses ; they 



342 THE SPUTUM. 

are secreted in old cavities which have become lined with a granu- 
lation-membrane. 

Very important is the presence of small, cheesy particles, which are 
occasionally found at the bottom of the spit-cup. They vary in size 
from that of a millet-seed to that of a pea, and are observed espe- 
cially in the second and third stages of phthisis. Usually they con- 
tain tubercle bacilli in large numbers, and frequently also elastic 
tissue. Not to be confounded with these, are certain small, caseous 
masses which are at times expectorated by perfectly normal indi- 
viduals, and also by patients suffering from acute tonsillitis, ozsena, 
etc., and which probably come from the tonsils or mucous cysts. 
Formerly they were regarded as tubercles, and in hypochondriac 
individuals their expectoration may cause a great deal of anxiety. 
They are quite readily distinguished from the true caseous masses 
expectorated by phthisical individuals by the following character- 
istics : as a rule, they are expectorated unaccompanied by pus or 
even by mucus ; rubbed between the fingers they emit an extremely 
offensive odor, which is referable to the presence of fatty acids ; an 
examination for tubercle bacilli, moreover, will prove entirely nega- 
tive. Quite characteristic, furthermore, is the peculiar, finely floc- 
culent, granular appearance of the sputa seen after perforation of 
an empyema into the lungs through a small aperture, which is not 
followed by pneumothorax. 

Occasionally, as in putrid bronchitis, and gangrene of the lungs, 
and also in chronic bronchitis, ultimately leading to the formation 
of bronchiectatic cavities, an exquisite sedimentation is observed. 
Such sputa when collected in a conical glass present three distinct 
zones : the one at the bottom contains the cellular elements of the 
sputum, the second the pus-serum ; and the third or superficial 
layer consists of mucus and contains many air-bubbles. 

MACROSCOPICAL CONSTITUENTS OF SPUTA. 

Elastic Tissue. — Of macroscopical constituents which may be 
observed in sputa, there may be mentioned, first of all, the occur- 
rence of threads of elastic tissue and pulmonary parenchyma, which 
are seen in cases of phthisis, pulmonary abscess, and gangrene. As 
their ultimate recognition, however, largely depends upon a micro- 
scopical examination, this subject Avill be considered later on. 

Fibrinous Casts. — Fibrinous casts are observed especially in 
cases of croupous pneumonia (Fig. 79), immediately before or after 
resolution has taken place. They are seen also in cases of so-called 
fibrinous bronchitis (Fig. 80), and in diphtheria when the membrane 
has extended into the finest ramifications of the bronchi. These 
casts may vary in size from 15 cm. in length by several milli- 
meters in thickness to fragments which measure only from 0.5 to 3 



MACROSCOPICAL CONSTITUENTS OF SPUTA 



343 



cm. in length. The fibrinous casts observed in cases of pneumonia, 
usually from the third to the seventh day, are of the latter size or 
even smaller, being derived from the ultimate twigs of the finest 
bronchioles. Those found in the rather rare disease, fibrinous bron- 
chitis, stand between these two in size, being casts of the smaller and 
medium-sized bronchi. Attention is usually attracted to the presence 
of such casts by their white color ; often, however, they are yellow- 
ish brown or reddish yellow, owing to the presence of blood-coloring 
matter which has become deposited upon the casts ; at other times 
they are enveloped in mucus, when their recognition may become 



Fig. 79. 




Fibrinous coagulum from a case of croupous pneumonia. (Bizzozero.) 

quite difficult. Such casts are fairly firm ; they branch dicho- 
tomously, usually 6 to 10 times. The larger branches contain a 
lumen, while the smallest twigs are solid. Microscopically they 
may be shown to consist of a large number of fibres, which are 
arranged longitudinally or in a net-like manner, and contain blood- 
corpuscles and epithelial cells in their meshes. When treated with 
Weigert's fibrin-stain, they are sometimes beautifully resolved ; at 
other times the fibrin reaction is not nearly so marked as one would 
expect. The individual casts consist of a variable number of laminae 
arranged concentrically, those contained in the centre being much 
folded and involuted. Most of the branches are cylindrical : some 



344 THE SPUTUM. 

of the larger ones are flat. Charcot-Leyden crystals have at times 
been observed in these formations. 

Fig. 80. 




Expectorated cast, from a case of fibrinous bronchitis. Three-fourths natural size. 
Drawn from fresh specimen, (After Bettmann.) 

Whenever it is desired to examine sputa for casts, it is best to 
pick out particles that look promising, upon a dark or light surface, 
and then to shake them out in water. For such purposes Kronig's 
sputum -plate can be recommended. 

Literature. — M. Bettmann, Am. Jour. Med. Sci., 1902, vol. cxxiii. p. 304 (a full 
review of all cases in the literature up to 1902 is here given). 

Curschmann's Spirals. 1 — Q,uite distinct from the formations 
just described are the so-called spirals of Curschmann, which are 
observed especially in cases of true bronchial asthma, hut occur also 
in chronic bronchitis, and even in croupous pneumonia. Upon 

1 Leyden, Virchow's Archiv, 1872, vol. liv. p. 328. Curschmann, Deutsch Arch. f. 
klin. Med., 1883, vol. xxxii. p. 1, and vol. xxxvi. p. 578. v. Jaksch, Centralbl. f. klin. 
Med., 1883, vol. iv. p. 497. 



MACROSCOPICAL CONSTITUENTS OF SPUTA. 345 

careful examination they will be seen to consist of thick, yellowish- 
white masses, which exhibit a spirally twisted appearance, and are 
characterized, moreover, by their more solid consistence and light 
color. On microscopical examination they are seen to be composed 
of a spirally twisted network of extremely delicate fibrils, containing 
epithelial cells and numerous leucocytes ; the latter are almost all 
of the eosinophilic variety. 1 Usually, but not invariably, Charcot- 
Leyden crystals also are seen. 2 The spirally twisted mass is found 
to be wound around a central, very light and clear thread, which 
usually has a zigzag course (Fig. 81). 

Other formations, probably mere varieties of those just described, 
have also been observed, in which the central thread is absent or in 
which the spiral arrangement is deficient. The spiral form, how- 
ever, with the central thread, must be considered as the most char- 
acteristic. Their length and breadth may vary a great deal, but 
rarely exceed 1 to 1.5 cm. Their occurrence seems always to indi- 
cate a desquamative catarrh of the bronchi and alveoli, but practi- 
cally nothing is known concerning their formation. If in a given 
case the diagnosis rests between true bronchial and what may be 
termed reflex asthma, the presence of these formations points to the 
existence of the former disease. Chemically, the spirally wound 






A Curschmann spiral from a case of true bronchial asthma. 

mass seems to consist of a mucinous substance, while the central 
thread is possibly of fibrinous origin. 

Charcot-Leyden crystals (Fig. 82), which are usually absent at 
the beginning of an attack of asthma, at which time only the spirals 
are observed, may be seen to develop from the spirals when these 
are kept for several days. They will be considered later in studying 
the chemistry of the sputum. 

Echinococcus Membranes. — Echinococcus membranes come 

1 Schmidt, Zeit. f. klin. Med., 1892, vol. xx. p. 92. v. Noorden, Ibid., p. 98. 

2 Ley den, loc. cit. 



346 



THE SPUTUM. 



from a perforating cyst of the liver, kidney, or lung. They consti- 
tute rather thick, and at the same time tough, pieces of membrane 
(Fig. 83) ; occasionally entire sacs are seen, of the color of white 
porcelain, in sections of which it is possible to make out a fibrillated 
structure. The disease is rare in this country. 



Fig. 82. 



Fig. 83. 





Charcot-Leyclen crystals. (Scheube.) 



Wall of a hydatid cast, showing 
the laminated structure; not mag- 
nified. (Davaine.) 



Concretions. — Still rarer is the expectoration of concretions which 
have formed in dilated portions of the bronchi or in tubercular 
cavities, or of calcified bronchial glands that have found their way 
into the lungs. Curious examples of the occurrence of such con- 
cretions have been reported. Andral thus cites a case of phthisis 
in which within eight months as many as 200 stones were expec- 
torated, and Portal mentions a case in which 500 were thus expelled. 1 

Foreign Bodies. — Foreign bodies which have accidentally entered 
the air-passages and have remained there for a long time may also 
be found in the sputum. Heyfelder mentions a case in which a 
man coughed up a wooden cigar-holder with pus and blood after 
eleven and a half years. 

MICROSCOPICAL EXAMINATION. 

Under this heading it is necessary to consider leucocytes, red 
blood-corpuscles, epithelial cells, elastic fibres, corpora amylacea, 
parasites, and crystals. 

Leucocytes. — Leucocytes, usually polynuclear in character, are 
found in every sputum in considerable numbers, imbedded in a 
homogeneous, more or less tenacious material. At times they appear 
very granular, containing fat-droplets, or granules of pigment, such 
as carbon or hematoidin. Their number varies considerably, being 
naturally greatest in cases of perforating abscess, empyema, putrid 
bronchitis, etc. 



1 L. W. Atlee, " Bronchial Concretions," Am. Jour. Med. Sci., 1901, vol. cxxii. p. 49. 
Fiessinger, " Oalcule pulmonaire," Jour, de Med., 1902, No. 29. 






jS? 



PLATE XVI. 



■m 

m 






"*§i^ 



•aKti* • ...■••••••.- ..^•^.•••«k5r-- ,5Sk i - • V. 












*&;; 

...: 



.-.- ? 

•% 



Sputum from a ease of Bronchial Asthma, showing large num- 
bers of Eosinophilic Leucocytes and Free Granules. 

It will be noted that the leucocytes are all mononuclear. (Eye-piece i, objective 1-8, Bausch and Lomb. 



MICROSCOPICAL EXAMINATION. 347 

While the leucocytes which usually are fouud in the sputum are 
of the neutrophilic variety, eosinophiles may also be observed, and 
especially in asthmatic sputa, in which they often predominate. 
Free eosinophilic granules are then also seen, and I have repeatedly 
observed specimens in which the spirals (see above) were literally 
covered with these granules (Plate XVI.). The presence of eosino- 
philic leucocytes is, however, not characteristic of the sputa of 
bronchial asthma, as they may be met with in other diseases as 
well. Teichmuller has pointed out that they are present in a large 
percentage of tubercular cases, and may be found months before 
tubercle bacilli can be demonstrated. He regards their occurrence 
as evidence of a defensive struggle on the part of the body, which 
is most evident in fairly strong individuals. In recovery a gradual 
increase in their number is always noticeable, and a diminution, 
Teichmiiller thinks, is indicative of a relapse, or, if the diminution 
occurs rapidly, of florid consumption. These statements, however, 
lack confirmation and are probably too dogmatic. Ott, Fuchs, Bett- 
mann, Turban, and Cohn, in fact, deny the prognostic siguificance 
of the eosinophilic cells in cases of phthisis ; and Cohn states as 
the result of an examination of 100 cases, many of which were 
comparatively early, that the occurrence of eosinophilic leucocytes 
is fairly uncommon in tubercular sputa. 1 

Stadelmann also states that he has been unable to verify Teich- 
miiller's observations. On the other hand, he has been able to 
confirm the observation which has been repeatedly made, that large 
numbers of eosinophilic cells appear in the sputum following hae- 
moptysis. Teichmuller has also described an " eosinophilic " bron- 
chitis, which is said to differ from other forms of the disease in the 
abundance of eosinophilic cells which are encountered. The sputum 
in such cases is described as transparent, mucoid, and loose, with 
yellow purulent admixtures. It is said to be markedly different 
from the tough, thick sputa of bronchial asthma. Typical spirals 
are absent, but rudimentary forms may be encountered. Charcot- 
Leyden crystals are present. 2 

Griinwald 3 states that in the sputa of the most diverse diseases 
cells are met with which contain a hypoeosinophilic granulation, and 
that the granules in question may also occur outside of the cells in 
the absence of evidence of special cell-destruction. These gran- 
ules, in contradistinction to the true eosinophilic cells, lose their 
color on treating with an acid, and readily take up the blue stain on 

1 Discussion on tuberculosis, Deutsch. med. Woch., 1901, V. B. p. 210. 

2 Teichmuller, " Die eosinophile Bronchitis," Deutsch. Arch. f. klin. Med., vol. 
lxiii. p. 444. See, also, K. Schonbrod, Ueber den gegemvartigen Stand der Beurthei- 
lung der eosinophilen Zellen im Blute und im Sputum, Inaug. Diss., Erlangen. 1895. 
A. Hein. Ueber das Vorkommen eosinophiler Zellen im Sputum, Inaug. Diss., Erlan- 
gen, 1894. 

8 L. Griinwald, "Studien iiber d. Zellen im Answurf," etc.. Virchow's Archiv, 
1899, vol. clviii. p. 297. 



348 THE SPUTUM. 

subsequent staining with methylene-blue. Grunwald states, hew- 
ever, that a sharp line of distinction does not exist between the two 
varieties of granules, and that intermediary conditions exist, as also 
transitions between oxyphilic and basophilic granules in the nature 
of an amphophilic granulation. 

To demonstrate eosinophilic leucocytes in the sputum, smears are 
made as usual, slightly fixed by drawing through the flame of a 
burner, and stained for two minutes in a 0.5 per cent, dilute alcoholic 
solution of eosin. The preparations are then immersed in 50 per cent, 
alcohol to the point of decolonization, when they are counterstained 
with methylene-blue, briefly washed with water, and dried. The 
eosinophilic granules and the red cells in part hold the eosin dye. 

Basophilic leucocytes have also been observed in the sputa. 

Red Blood-corpuscles.— The presence of red blood-corpuscles in 
small numbers does not, by any means, indicate serious pulmonary or 
cardiac disease, as they may be found in almost any sputum, and 
especially in that of individuals who smoke much or live in a smoky 
atmosphere ; they are, without doubt, derived from the catarrhally 
inflamed bronchial or tracheal mucosa. Whenever they occur in 
large numbers, however, their presence becomes important. They 
may be observed in acute bronchitis, pneumonia, oedema of the 
lungs, bronchiectasis, abscess, gangrene — in fact, in all pulmonary 
diseases. Their occurrence is most important in phthisis, and is, in 
fact, one of the most constant symptoms of the disease. 

The form of the red corpuscles will depend upon the length of 
time they have remained in the lungs, and all gradations from the 
typical red corpuscle to its shadow, or even fragments, may thus be 
observed. In pneumonia the microscopical examination may at 
times be disappointing, the appearance of the sputum suggesting 
that red corpuscles in large numbers are present, while, as a matter 
of fact, they are almost all destroyed, the color being due to altered 
pigment. It may even be necessary at times to depend upon chemi- 
cal methods to clear up any doubt as to the source of the color of 
the sputum. It should always be remembered that the presence of 
blood-pigment is not always indicated by a red color, but that it may 
also assume a golden-yellow or even a greenish tinge, owing to cer- 
tain chemical changes which have taken place. The golden-yellow 
and the grass-green sputa observed in cases of pneumonia during 
convalescence belong to this class. 

To demonstrate the presence of traces of blood in the sputum, 
Donogany's method, or that of Muller and Weber, may be con- 
veniently employed. With the former method the sputum is first 
boiled with a 20 per cent, solution of sodium hydrate (see page 262). 

Epithelial Cells. — Epithelial cells are found in practically every 
sputum. Cylindrical epithelial cells, providing they do not come 
from the nose, indicate in a general way an inflammatory condition 



MICROSCOPICA L EXA MINA TION. 



349 



of the lower larynx, trachea, or bronchi. They are not of much 
importance, however, as their form is usually so much altered that 
it is often difficult to recognize them ; they may thus become poly- 
hedral, cuboidal, or even round, and can then hardly be distinguished 
from leucocytes. Actively moving cilia may be found only in per- 
fectly fresh sputa, immediately after being expectorated. If ciliated 
epithelial cells can be definitely recognized in a sputum, it may be 
inferred that we are dealing with a pathological condition of an acute 
nature, providing, of course, they did not come from the nose. 

Formerly much importance was attached to the so-called alveolar 
epithelial cells (Fig. 84) as an aid in diagnosis. Buhl thus imagined 
these, particularly when undergoing fatty or myelin degeneration, 
to be absolutely pathognomonic of pulmonary disease, and especially 
of that form of pneumonia which has been termed essential idio- 
pathic desquamative pneumonia. Bizzozero, however, as well as 

Fig. 84. 



o?o 




Epithelium, leucocytes, and crystals of the sputum. (Eye-piece III., objective 8 A, Reich- 
ert.) a, a', a", alveolar epithelium ; b, myelin forms ; c, ciliated epithelium ; d, crystals of 
calcium carbonate : e, hsematoidin crystals and masses ; /,/,/, white blood-corpuscles ; g, red 
blood-corpuscles ; h, squamous epithelium, (v. Jaksch.) 

others, has shown that these cells not only occur in almost every 
known pulmonary disease, but that they are present also in the so- 
called "normal" expectoration which at times is obtained upon 
making a very forcible expiration. 

Bizzozero x describes these cells as round, oval, or polygonal bodies, 
varying in size from 20 p. to 50 //. They may contain one, two, or 
three oval nuclei, which are rather small and provided with nucleoli. 
Usually the latter are hidden beneath numerous granules. Some of 
these granules are albuminous, but most of them are either pigment- 
granules, fatty granules, or myelin granules. The myelin granules 
were first discovered by Virchow 2 in 1854, and termed myelin gran- 
ules on account of their resemblance to mashed nerve-matter. They 
are distinguished from the other forms by their clear, pale, color- 

1 Bizzozero, Microscopie clinique, 2d ed. Francaise, Paris, 1885. 

2 Virchow, Virchow's Archiv, 1854, vol. vi. p. 562. 



350 THE SPUTUM. 

less appearance, and the fact that at times fine concentric striations 
can be detected. These forms may be round, but more often they 
are irregular. At times fatty, myelin, and pigment-granules may 
be seen in one and the same cell. Possibly they are derived from 
the pulmonary alveoli, but this is still an open question. Chemi- 
cally, the myelin droplets have been shown to contain a considerable 
amount of protagon, besides traces of lecithin and cholesterin. 1 

The sputa of chronic bronchitis referable to heart disease are 
characterized by the presence of so-called heart disease cells. These 
are alveolar epithelial cells containing numerous hsematoidin gran- 
ules (Plate XVIL, Fig. 3). They appear to be most numerous 
in cases of mitral disease, but may also occur in congestive affec- 
tions of the broncho-pulmonary apparatus, even with the heart 
intact. 2 

Liver-cells may at times be observed in the sputa in cases of liver- 
abscess, and are easily recognized by their characteristic form. 

Elastic Tissue. — Much more important from a clinical stand- 
point are the elastic fibres and shreds of elastic tissue which may 
be found in sputa. They vary much in length and breadth, and 
are provided with a double, undulating contour ; they are usually 
curled at their ends. Very often they exhibit an alveolar arrange- 
ment (Fig. 85), which at once determines their origin. 

Fig. 85. 




Elastic fibres in the sputum. (Eye-piece III., objective 8 A, Reichert.) (v. Jaksch.) 

Whenever present, elastic tissue is an absolute indication that a 
destructive process is going on in the lungs. It is found in cases of 
abscess of the lungs, bronchiectasis, occasionally in pneumonia, pul- 
monary gangrene and infarct, and, most important of all, in phthisis, 
in which it is said to be present in 90 per cent, of all cases. This 

1 A. Schmidt, "Ueber Herkunft u. chem. Natur d. Myelinformen d. Sputums," 
Berlin, klin. Woch., 1898, p. 73. See, also, Zoja, Maly's Jahresberichte, vol. xxiv. 
p. 694. 

2 K. C. Regolo, Gaz. d. Ospedali, Milauo, vol. xxii. No. 135, 



PLATE XVII 

FIG. 1. 



■•■' f '- 


> 


"^ 


".'« \\ 



* 



Tuberculous Sputum Stained, by Gabbett's Method. The Turberele Bacilli 
are seen as Red Rods, all else is Stained Blue. (Abbott.) 

FIG. 2. 




L. SCHMIDT, FEC. 



The Diploeoeeus Pneumoniae, Stained with Methylene Blue and Fuehsin 
as a Counterstain. Taken from the Sputum of a Case of 
Acute Croupous Pneumonia. 

FIG. S. 









4 ^ 



JO; T# * 



<*&' 



Heart-Disease Cells, showing Alveolar Epithelial Cells, Loaded Down 
with Granules of Hsematin. 



MICROSCOPICAL EXAMINATION. 351 

percentage, which was obtained by Dettweiler and Setzer in 1878, 
is unquestionably too high in comparison to what is seen to-day 
when the diagnosis of tuberculosis is after all made much earlier. 
In gangrene of the lung elastic tissue is usually not found ; this is 
probably owing to its destruction by a ferment, as suggested by 
Traube. 

In every case it is necessary to determine whether the elastic 
tissue may not be owing to the presence of animal food in the 
sputum, and it may hence be stated as a rule that it can only 
be regarded as absolutely characteristic when showing the alveolar 
arrangement. 

In order to demonstrate the presence of elastic tissue in the 
sputum the following method, in use at the Johns Hopkins Hospital, 
is very convenient : a small amount of the thick, purulent portion 
of the sputum is pressed into a thin layer between two pieces of 
plain window-glass, 15 by 15 cm. and 10 by 10 cm. The particles 
of elastic tissue appear on a black background as grayish-yellow 
spots, and can be examined in situ under a low power. Or, the 
upper piece of glass is slid off till the piece of tissue is uncovered, 
when it is picked out and examined on a slide, first with a low and 
then with a higher power. At first there will be some difficulty in 
distinguishing with the naked eye between elastic fibres and particles 
of bread, or milk globules, or collections of epithelium and debris, 
but with practice such mistakes are rarely made, and the microscope 
always reveals the difference. 

If only very little elastic tissue is present, it is necessary to 
examine large quantities of sputum with a moderately low power, 
and best after the addition of a solution of sodium hydrate. The 
sputum is boiled with a 10 per cent, solution of the reagent, an 
equal volume being added ; the boiling is continued until a homo- 
geneous solution has been obtained ; after dilution with four times 
its volume of water it is allowed to settle for twenty-four hours. 
The centrifugal machine will here be found of great assistance. 
The elastic tissue fibres are found in the sediment. 

To stain elastic tissue, Michaelis suggests the following method : 
suspected bits of sputum are spread upon a slide in a thin layer, dried, 
and then placed for one-half hour in a jar containing Weigert's solu- 
tion. The specimen is then washed with water, decolorized in acid 
alcohol (containing 3 per cent, of hydrochloric acid), dried, covered 
with a thin layer of oil of cedar, and examined without a cover-glass 
with a low power ; the elastic fibres are stained a dark violet. 

Weigerfs Elastic Tissue Stain. — This is prepared as follows : 
200 c.c. of an aqueous solution of fuchsin and resorcin, containing 
1 and 2 per cent, of the ingredients, respectively, are boiled in a 
porcelain dish. When the boiling-point is reached 25 c.c. of liquor 
ferri sesquichloridi (Ph. G. III.) are added. While stirring the 



352 THE SPUTUM. 

solution is boiled for from two to five minutes longer. It is then 
allowed to cool ; the precipitate is collected on a filter, dried, and 
boiled in 200 c.c. of 94 per cent, alcohol while stirring. On cool- 
ing, alcohol is added to the 200 c.c. mark, when the solution is 
treated with 4 c.c. of hydrochloric acid, and is ready for use. 

May 1 recommends the following method of demonstrating the 
presence of elastic tissue in sputum : The material in question is 
heated on a boiling water-bath with an equal volume of a 10 per 
cent, solution of sodium hydrate until it has all apparently dissolved. 
The mixture is then centrifugal ized and the supernatant fluid 
decanted. The sediment is treated with about 2 c.c. of an orcein 
solution prepared according to the formula of Unna-Tanzer, viz., 
orcein, 1 gramme ; absolute alcohol, 80 c.c. ; distilled water, 40 c.c. ; 
concentrated hydrochloric acid, 40 drops. On adding the stain, 
owing to the remaining alkali, the color turns violet ; a few drops 
(3—5) of hydrochloric acid are added until the original color of the 
stain returns. The tube is then placed for from two to five minutes 
in boiling water, after which acid alcohol (concentrated hydrochloric 
acid, 5 c.c. ; 95 per cent, alcohol, 1000 c.c. ; distilled water, 250 c.c.) 
is added to decolorize. The mixture is again centrifugalized and 
the sediment washed once or twice more with the acid alcohol by 
centrifugation and decantation. The sediment is then examined 
directly, when the elastic tissue fibres may be recognized by their 
more or less intense brownish-violet color. 

Animal Parasites. 

Taenia Echinococcus. — Portions of echinococcus cysts, viz., 
pieces of membrane (Fig. 84) and booklets (Fig. 86), are occasion- 
ally seen when the parasite has lodged in the lungs or in the neigh- 
boring organs. The disease is not common in this country. Lyon 2 
has collected a total of 241 cases in the United States and Canada 
up to July 1, 1901. 91 per cent, occurred in foreigners. In 
Canada a large proportion is referable to the Icelandic immigrants 
in Manitoba. 

The adult parasite (Fig. 87) (v. Siebold) is found in the intestinal 
canal of the dog, the dingo, the jackal, the wolf, etc. The larval 
form, Echinococcus polymo?-phus, develops in cattle, sheep, and 
swine, and is also found in man. The parasite, in fact, is the most 
dangerous animal parasite which is encountered in the human being. 
If the eggs of the parasite are .introduced into the digestive tract 
of man, the embryos may make their way into the lungs, liver, or 
other organs, and there give rise to the formation of cysts, which 
are often of enormous size. The body of the adult animal is from 

J E. May, Deutsch. Arch. f. klin. Med., 1900, vol. lxviii. p. 427. 
2 1. P. Lyon, N. Y. State Jour. Med., Oct., 1902. 



MICROSCOPICAL EXAMINATION. 



353 



4 to 5 mm. long, with only 3 or 4 segments, the largest of which 
may measure 0.6 mm. in length by 2 mm. in breadth. On the 
head there are from 28 to 50 hooklets (see Fig. 87). 1 



Fig. 



<$^ 



f 



3 * f 

Hooklets from Taenia echinococcus. X 350. 

Trichomonad.es have at times been observed in cases of gangrene 
of the lung, and in the pus removed post mortem from lung-cavities. 
They are identical with the Trichomonas vaginalis of Donne. 




Human echinococcus. (From Finlayson, after Davaine.) A, a group of echinococci, still 
adhering to the germinal membrane by their pedicles. X 40. B, an echinococcus with head 
invaginated in tne body. X 107. C, the same compressed, showing suckers and hooks of the 
retracted head. D, echinococcus with head protruded. E, crown of hooks, showing the two 
circles. X 350. 

Amoeba Coli. — The presence of this parasite is most important, as 
the diagnosis of hepatic abscess with perforation into the lung may 
be made in every instance in which the organism is encountered in 
the sputa (see Feces). 2 

Distoma Pulmonale. — A form of pulmonary disease closely sim- 

1 Hydatid disease in man : Neisser, Die Echinococcen-Krankheit, 1877, Berlin. 
Davaine, Traite des Entozoaires et des Maladies vermineuses, Paris, 1877, 2d ed. 

2 C. E. Simon, Johns Hopkins Hosp. Bull., Nov., 1890. 

23 



354 THE SPUTUM. 

ulating phthisis is very common in Japan, and has been shown to be 
referable to the presence of a parasite in the lungs, Distoma pulmo- 
nale Balz (syn., Distoma Westermanni (Kerbert), Distoma Ringeri 
(Cobbold)). The parasite is 8 to 10 mm. long, 4 to 6 mm. wide, 
rounded very markedly in front, less so posteriorly. The color 
during life is a reddish-brown. The two sucking disks are nearly 
equal in size. The ova are brown, with a thin shell and lidded. 
They measure from 80 to 100// in length and 40 to 60 fi in breadth. 
The worm and its ova are found in the sputum. If the sputum is 
shaken in water and the water renewed from time to time, in the 
course of a month or six weeks (according to the temperature) a 
ciliated embryo is developed in each ovum. When the ovum is 
mature, on placing it on a slide and exercising slight pressure on the 
cover-glass, the operculum will be forced back, and the embryo will 
emerge and at once begin to swim and gyrate in the water (Manson). 
Outside of Japan the parasite has been found in Corea and Formosa. 
In the United States it has been found in the cat and in the dog ; in 
the human being one case at least, occurring in a Japanese student, 
has been reported. Many Charcot-Leyden crystals are found in the 
sputum at the same time. 

Literature. — C. D. Stiles, " Distoma Westermanni," Johns Hopkins Hosp. Bull., 
1894, p. 57. Brown, Die thierischen Parasiten, etc., Stuber, Wurzburg, 1895. 

Distoma Haematobium. — Manson found the ova of a species of 
Distoma hsematobium in the bloody expectoration of a Chinaman 
who had lived for some time on the island of Formosa. 

Vegetable Parasites. 

Pathogenic Organisms. — The Tubercle Bacillus. — The most im- 
portant vegetable parasite met with in the sputa is the bacillus of 
tuberculosis. The history of the discovery of this organism, and the 
theories which were held before its pathogenic importance was estab- 
lished, cannot be considered here. Suffice it to say that the study 
of bacteriology has given no other discovery of equal importance 
from a clinical point of view. How primitive and wholly inadequate 
were the means formerly employed in making the diagnosis of this, 
the most formidable disease of modern times ! The presence or 
absence of elastic tissue in the sputa was practically all that 
physicians had to guide them beyond the history of the patient 
and the results of a physical examination. The demonstration of 
elastic tissue, however, as has been pointed out, merely indicates the 
existence of a destructive process in the lungs. Under such condi- 
tions it was of necessity impossible to diagnose tubercular disease 
in its incipiency. It is true that cases are occasionally observed in 
which tubercle bacilli are never present in the sputa, and are only 
discovered post mortem. Such cases, however, are extremely rare, 



MICROSCOPICAL EXAMINATION. 355 

and do not in the least detract from the importance which attaches 
to careful and repeated examinations of the sputa in all doubtful 
cases. 

From a macroscopical examination it is impossible to decide 
whether or not a particular sputum is of tubercular origin. At times 
a sputum may have a suspicious appearance, but it is never possible 
to speak with certainty from simple inspection, as a mucoid sputum 
may contain tubercle bacilli in large numbers, while a muco-purulent 
sputum may be entirely free from them, and vice versa. Reliance 
should, hence, only be placed upon a careful microscopical examina- 
tion. When found, their presence is, of course, pathognomonic. A 
negative result, however, does not exclude the existence of tuber- 
cular disease. The possibility that they may be altogether absent 
from the sputum has been mentioned. In some instances they may 
be present at times and absent at others. In all cases in which the 
existence of phthisis is suspected, it is imperative to make use of 
every device which may aid in its detection. In this connection, 
I wish to insist upon the method of " growing the bacilli," as it 
were, in the warm chamber for from twenty-four to forty-eight 
hours, and then re-examining the sputa in doubtful cases, as 
JSTuttall l demonstrated beyond a doubt that the tubercle bacillus 
will multiply in the sputum itself at a certain temperature. The 
value of this observation is obvious, and I have repeatedly been 
able to demonstrate their presence in this manner when it was 
impossible to detect them in the fresh sputum. The centrifugal 
machine in such cases is also useful and yields valuable results, the 
probabilities of finding the bacilli when present in small number 
being very much increased. 

In the examination of tubercular sputa the fine caseous particles 
previously described (page 342) should be carefully sought for, as 
they contain the largest number of bacilli. In their absence reliance 
should be placed upon the examination of a large number of prepa- 
rations. 

If but few bacilli are present, the following procedure may also 
be employed : about 100 c.c. of sputum are boiled with double the 
amount of water, to which from six to eight drops of a 10 per cent, 
solution of sodium hydrate have been added, until a homogeneous 
solution has been obtained, water being added from time to time to 
allow for evaporation. The mixture is then centrifugated or set 
aside for twenty-four to forty-eight hours and examined for tubercle 
bacilli and elastic tissue. Or, the following procedure, suggested by 
d ? Arrigo and Stampacchia, may be employed : Four or five sputum 
masses are placed in a test-tube and covered with Ranvier's acid 
alcohol (70 per cent, alcohol, containing 1 per cent, of concentrated 
hydrochloric acid), so that this fills about two-thirds of the tube. 

1 Nuttall, Johns Hopkins Hosp. Bull., 1891. 



356 THE SPUTUM. 

The mixture is well shaken and is kept, stoppered with cotton, for 
twenty-four hours at 37° C. or for three hours at 50° C. The acid 
alcohol destroys the mucus and fixes the cells and bacilli, which sink 
to the bottom. It is claimed that in a sediment prepared in this 
manner it is possible to demonstrate the tubercle bacilli even after 
several years. 

If, notwithstanding the fact that all due precautions have been 
taken, no bacilli can be demonstrated in the sputum, and the clinical 
history and the physical signs are indefinite or negative, the proba- 
bilities are that we are dealing with a benign process. From an 
examination of the sputa alone in such cases it is utterly impossible 
to reach a definite conclusion. When the amount of sputum, more- 
over, is small and contains but little pus, the absence of tubercle 
bacilli in doubtful cases is less suggestive of the absence of tuber- 
cular disease than in cases in which the sputum is more abundant 
and mucopurulent. 

Only two bacilli are likely to be mistaken for the tubercle ba- 
cillus, viz., the bacillus of leprosy and the smegma bacillus. All 
three are characterized by the difficulty with which they take up 
basic dyes, and the great tenacity with which these are retained 
when once stained, even upon treatment with mineral acids. This 
peculiarity has been quite generally referred to the presence of fat 
in the bacilli, but it appears from more recent researches that the 
chitin or chitinous substances in the bodies of the tubercle bacilli 
are primarily concerned in the reaction (Helbing 1 ). Sata, 2 moreover, 
has shown that other bacteria, such as the anthrax bacillus, the 
bacillus of glanders, the staphylococcus aureus, etc., give a fat 
reaction which is as intense as that of the tubercle bacillus, while 
these organisms are not in the least resistant to the action of acids 
when stained. 

That confusion should arise in the differentiation between the 
tubercle bacillus and the bacillus of leprosy is very unlikely. More 
important is the smegma bacillus, which is now known to occur at 
times upon the tonsils, the tongue, and in the tartar of the teeth of 
perfectly healthy individuals. In sputum coming from the lungs 
it has been observed by Pappenheim, 3 Frankel, 4 and others. To 
Pappenheim we are indebted for a method by which we are enabled 
to differentiate such cases from tuberculosis. This is essentially 
based upon the greater ease and rapidity with which the smegma 
bacillus is decolorized by means of fluorescem-alcohol, as compared 

1 C. Helbing, " Erklarungsversuch f. d. specifische Farbbarkeitd. Tuberkelbacillen," 
Deutsch. med. Woch., 1900, V. B. p. 133. 

2 Sata, " Ueber d. Fettbildung durch verschiedene Bakterien," etc., Centralbl. f. 
allg. Path. u. path. Anat., 1900, Nos. 3, 4. 

3 A. Pappenheim, " Befund v. Smegmabacillen im menschlichen Lungenauswurf. " 
Berlin, klin. Woch., 1898, No. 37. 

4 A. Frankel, " Einige Bemerkungen iiber d. Vorkommen v. Smegmabacillen im 
Sputum," Ibid., 1898, p. 880. 



MICROSCOPICAL EXAMINATION. 357 

with the tubercle bacillus. As the other methods which have hitherto 
beeu in use in the clinical laboratory do not permit of differentiation 
between the two organisms, I have given Pappenheim's method the 
first place, but have retained the others also. They may be em- 
ployed as heretofore, unless special reasons exist for eliminating the 
smegma bacillus, the occurrence of which in the sputum must after 
all be regarded as a medical curiosity. In the examination of 
urinary deposits, however, in which the smegma bacillus is far more 
commonly seen, these older methods are not applicable (see Urine). 

Methods of Staining the Tubercle Bacillus. — 1. Pappen- 
heim's Method. 1 — A drop of the sputum — or, if the cheesy particles 
described above, are present, one of these — is spread in a thin layer 
between two cover-glasses. These are then drawn apart, dried in 
the air, and fixed by being passed three times through the flame of 
a Bunsen burner or an alcohol lamp. Larger quantities of the 
sputum may also be employed, and are spread upon slides and 
examined in the same manner, a drop of immersion oil being 
placed directly upon the dried and stained preparation. The speci- 
mens are covered with a few drops of carbol-fuchsin solution and 
heated to the boiling-point. The solution is composed of 1 part of 
fuchsin, 100 parts of a 5 per cent, solution of carbolic acid and 10 
parts of absolute alcohol. The excess of the staining fluid is drained 
off, when the preparations are immersed from three to five times in 
Pappenheim's solution, care being taken to let the fluid drain off 
slowly after each immersion. The reagent consists of 1 part of 
corallin (rosolic acid) in 100 parts of absolute alcohol, to which 
methylene-blue is added to saturation. This mixture is further 
treated with 20 parts of glycerin, and is then ready for use. The 
specimens are finally washed in water, dried between filter-paper, 
and mounted in balsam or oil of cedar. A -^ oil immersion lens is 
very convenient, but not a necessity, as the organisms are seen quite 
readily with lower powers, such as Zeiss' DD, Leitz' 7, or Bausch 
and tomb's |or{, with a correspondingly high eye-piece. 

2. Gabbet's Method. — The dried and fixed preparations are covered 
for two minutes with the carbol-fuchsin solution described above, 
and are immediately transferred, without washing, to a solution com- 
posed of 2 parts of methylene-blue in 100 parts of a 25 per cent, 
solution of sulphuric acid, in which they remain one minute. They 
are then washed in water and mounted. 

Instead of staining with the carbol-fuchsin by the cold method, as 
just described, one can also use heat. To this end, the cover-glass 
or slide specimen is covered with the fuchsin solution and held over 
a small flame, so that the stain just barely simmers. The heating is 
continued for a minute or two, new reagent being added if evapo- 
ration should proceed too far. The process is then continued as 

1 Pappenheim, loc. cit. 



358 THE SPUTUM. 

described. Some investigators prefer to immerse the specimens in 
the carbol-fuchsin solution for twenty-four hours, but there is no 
material advantage to be gained in this way. 

It has recently been suggested by Pagani 1 to use lactic acid instead 
of sulphuric acid, in order to avoid a too energetic decolorization. 
He claims that excellent results are obtained if the second solution 
of Gabbet is replaced by one of the following formula : water, 50 
c.c. ; alcohol, 50 c.c. ; lactic acid, 2.5 grammes; and methyl-blue to 
saturation. The cover-glass specimens or slides are immersed in this 
solution for from fifteen to twenty seconds while gently agitating. 

Gabbet's method of staining is very convenient, and is the one 
most generally employed. The smegma bacillus, however, is also 
stained. 2 

3. The Weigert-Ehrlieh Method. — Dried specimens are prepared, 
and stained for twenty-four hours with a solution of fuchsin in 
anilin-water, by floating upon the surface. The staining fluid is 
prepared as follows : 

A small test-tube full of water is shaken with about twenty drops 
of pure anilin oil (1 : 20), and after standing for a few minutes fil- 
tered through a moistened filter. To this solution a few drops of a 
concentrated alcoholic solution of fuchsin or of methyl-violet are 
added until the mixture becomes slightly cloudy — i. e., until a metal- 
lic lustre is noted on the surface. After twenty-four hours the 
preparations are washed with water in order to remove an excess of 
staining fluid, They are then immersed for several seconds in a 
dilute solution of nitric or hydrochloric acid (1 : 6, 1 : 3, or 1 : 2), and 
washed again with water or with absolute alcohol. At this time the 
specimens should have a faintly red or violet color. They are then 
dried between layers of filter-paper or in the air, and mounted as usual. 

If it is desired to use a counter-stain, Bismarck-brown, vesuvin, 
or methylene-blue in watery solutions may be used for the purpose. 
Into such a solution the specimen is placed after treatment with 
nitric acid and washing in water. It remains for about two min- 
utes, and is then washed, dried, and mounted as above. 

4. Ziehl-Neelseri' 's Method. — A mixture of 90 parts of a 5 per 
cent, solution of carbolic acid and 10 parts of a concentrated alco- 
holic solution of fuchsin is used. The procedure is the same as that 
described under the Weigert-Ehrlieh method. It is usually not 
necessary to stain the preparations for twenty-four hours, however, 
and as a rule it is sufficient to place a few drops of the staining 
fluid upon the preparation and to heat over the free flame as de- 
scribed when the specimen is decolorized as before. In this manner 
excellent results may be obtained in a few minutes. 

1 Pagani, Ref/in Centralb. f. Path. u. path. Anat., 1901, vol. xii. p. 323. 

2 Fr'ankel, Berlin, klin. Woch., 1884, vol. xxi. p. 195; and Deutsch. med. Woch., 
1887, vol. xvii. p. 552. 



MICROSCOPICAL EXAMINATION. 359 

Stained according to one of these methods, the bacilli appear as 
rods, measuring about 1.5-3.5 [x in length by 0.2 ti in breadth 
(Plate XVII., Fig. 1). Much larger specimens may, however, also 
be seen, up to 11 it in length. The shortest forms are commonly 
straight; the common types are usually slightly curved. They 
may occur joined in chains of two or three, and branching forms 
have also been observed. Occasionally one may see a couple of 
organisms, each bent to a crescent, linked in the form of the letter 
S. Very commonly they are beaded, and it is possible to make out 
from 1 to 8 clear spaces in an organism which are separated by 
round or rod-shaped granules, which are deeply stained and appear 
to lie in a lightly staining capsule. The small hyaline bodies were 
once regarded as spores, but it is more likely that they are vacuoles. 
Sometimes bacilli are seen which have club- or knob-shaped swell- 
ings at the extremities. These enlargenents likewise have been 
viewed by some as spores, while others look upon them as products 
of degeneration. When present in large numbers, they are often seen 
in clumps, as though the bacilli had been agglutinated side by side, 
but in every specimen isolated organisms are also found scattered 
through the field ; or two and three are found together. 

Cultivation of the Tubercle Bacillus.- — The cultivation of the tuber- 
cle bacillus is best accomplished on blood-serum or glycerin-agar 
(agar with 6 per cent, of glycerin added) at a temperature of 37° 
or 38° C. Below 30° C. and at a temperature higher than 42° C. 
the organism does not grow. Primary inoculation from the tissue 
should be made on blood-serum, as the bacillus usually does not 
grow on glycerin-agar when this is inoculated directly from the 
tubercular focus. Subcultures, however, grow readily on glycerin- 
agar and more rapidly than on blood-serum. The individual colo- 
nies appear like small dry scales, which gradually coalesce and form 
a wrinkled film of a dull whitish color. Older cultures present a 
brownish or grayish-brownish color. An adequate idea may be 
formed of the growth of the organism after from two to three weeks. 
Sunlight rapidly kills the tubercle bacillus. 

The number of bacilli which may be found in a sputum varies 
greatly, and while in general it may be said that it is in direct 
ratio to the intensity of the disease, and may thus be considered of 
prognostic value, too much reliance should not be placed upon 
this statement, as in acute miliary tuberculosis, and in cases that 
have gone to the formation of cavities, the number may be small 
or they may be absent altogether. In an incipient case, on the other 
hand, in a little mucoid sputum the number may be large. If the 
number of bacilli steadily decreases in a series of examinations at 
intervals sufficiently long, the patient may be regarded as improv- 
ing, but here the constitutional symptoms and local signs give much 
more accurate information. 



360 THE SPUTUM. 

If on repeated examination large numbers of tubercle bacilli are 
found, the disease has in all probability advanced to cavitation 
(Brown). 

In tabulating the number of tubercle bacilli in reports one may 
adapt Gaffky's scheme, modified by L. Brown as follows (^ oil 
immersion ; ocular 1 ; B. & L.). 

1. Only 1—4 in a whole preparation. 

2. Only 1 bacillus on an average in many fields. 

3. Only 1 bacillus on an average in each field. 

4. 2-3 bacilli on an average to each field. 

5. 4-6 bacilli on an average to each field. 

6. 7—12 bacilli on an average to each field. 

7. 13-25 bacilli on an average to each field. 

8. About 50 bacilli on an average to each field. 

9. 100 or more bacilli on an average to each field. 
10. Enormous numbers on an average to each field. 

An attempt has been made to attach prognostic significance to 
form and grouping of the tubercle bacilli in the sputum. To judge 
from the experience gathered at Saranac, it appears that virulent 
and attenuated forms of tubercle bacilli possess practically the same 
morphology and that short bacilli usually represent a younger 
growth. Arrangement of the bacilli in clumps is more apt to be 
found in the severer cases, but may occur in all (Brown). 

Of the variations in number and form of the tubercle bacilli 
during treatment with Koch's tuberculin it is unnecessary to speak 
at this place, as the prognostic significance attaching to such varia- 
tions is questionable. 1 

The Diplococcus Pneumoniae. — In doubtful cases the sputum may 
be examined for the Diplococcus pneumonia?, and it may be accepted 
at the present time that its presence in a given case, providing that 
the clinical history and the physical signs point to a pneumonia, 
renders the diagnosis of acute croupous pneumonia very probable. 

Method. — Cover-glass specimens, prepared as indicated above, 
are placed for one or two minutes in a 1 per cent, solution of acetic 
acid ; they are then removed, the excess of acetic acid is drawn off 
by means of a pipette, when they are allowed to dry in the air ; they 
are subsequently placed for several seconds in saturated anilin- water 
and gentian- violet solution, washed in water, and examined. Rod- 
shaped diplococci (Plate XVII., Fig. 2), surrounded by a capsule, 
which latter is considered the characteristic feature of this organ- 
ism, will be seen in cases of acute croupous pneumonia. 2 

The bacillus of influenza has already been considered in Chapter 

1 F. Fischel, Unters. iiber d. Morphol. u. Biol. d. Tuberculose, Erregers, 1895. 
Gaffky, Mitth. aus d. Kais. Gesundh. Anz., vol. xi. p. 126; L. Brown, Jour. Am. Med. 
Assoc, 1903, vol. xl. p. 514. 

2 Frankel, Zeit. f. klin. Med., 1886, vol. ii. p. 437. Weichselbauni, Wien. med. 
Woch., 1886, vol. xxxix. pp. 1301, 1339, 1367. 



MICROSCOPICAL EXAMINATION. 361 

I. (page 122). In the sputum it is frequently associated with pyo- 
genic cocci and pneumococci. 

In whooping-cough protozoa have been observed by Deichler and 
Kurloff ; their observations have not been confirmed, however, and 
other observers attribute the disease to the presence of bacteria. 
Among these may be mentioned Affanasiew, Bitter, Czaplewski, 
Hensel, Koplik, and others. All these investigators claim to have 
isolated from the sputum of whooping-cough a micro-organism, 
which they regard as the cause of the disease. Whether or not 
Affanasiew' s bacillus is identical with Bitter's diplococcus and with 
the pole-bacillus of Czaplewski, Hensel, and Koplik 1 is, however, 
not clear. Koplik's organism is extremely minute, measuring from 
0.8 fi to 1.7/7. in length by 0.3 /j. to 0.4 fi in breadth. When 
stained with Loffler's blue it has a finely punctate appearance, like 
the diphtheria bacillus. In pure culture it is not decolorized by 
Gram's method. It is anaerobic as well as aerobic, and is apparently 
not motile. To isolate it from the sputum, it is best to obtain some 
of the grayish-white pellets which are expectorated during the con- 
vulsive stage. In these, small particles will be seen, resembling 
scales of dandruff. Such particles are isolated and planted first on 
hydrocele fluid, in order to obtain the crude culture. Later the 
organism may be grown in bouillon, on agar, gelatin, etc. On 
Loffler's serum a whitish growth is obtained which closely simulates 
that of the diphtheria bacillus. The organism is pathogenic for 
mice, particularly after intraperitoneal inoculation, but it does not 
produce whooping-cough in the lower animals. 

The Smegma Bacillus. — In a few isolated cases the smegma bacil- 
lus has been encountered in the sputum, and, as I have already 
stated, the same organism may normally be present in the saliva, 
the coating of the tongue, the tartar of the teeth, etc. Like the 
tubercle bacillus, it resists the decolorizing action of acids when 
once stained, and may hence be confounded with it unless special 
precautions are observed (page 357). 

Babinowitch 2 recently succeeded in cultivating from the spu- 
tum of a case of pulmonary gangrene an organism which is 
either identical with the smegma bacillus or closely allied to it ; 
she gives the following account of its cultural characteristics : on 
glycerin-agar, after twenty -four to forty-eight hours the organism 
forms grayish-white, lustrous colonies of the size of the head of a 
pin, which gradually coalesce to a whitish, cream-like coating. On 
further growth the lustre disappears, the surface appears dry, the 
coating becomes wrinkled and assumes a yellowish color. Still later, 

1 E. Czaplewski u. E. Hensel, "Bacterid. Untersuchungen bei Keuchhusten," 
Deutsch. rued. Woch., 1897, p. 586. H. Koplik, "The Bacteriology of Pertussis/' 
Johns Hopkins Hosp. Bull., 1898, p. 79. 

2 L. Rabinowitch, " Befund y. saurefesten tuberkelbacillenahnlicheu Bakterien bei 
Lungengangran," Deutsch. med. Woch., 1900, No. 16. 



362 THE SPUTUM. 

when kept at the temperature of the room it turns to a deep orange. 
The organism is non-motile. It occurs in the form of little rods, 
which in older cultures manifest a tendency to the formation of 
long threads. In gelatin stab-cultures small colonies appear along 
the line of the puncture, which are separated from each other. On 
the surface a thickish, white, lustrous coating develops, which gradu- 
ally turns orange. The gelatin is not liquefied. On potato the 
cultures form a moist, gray coating after two or three days. Bouil- 
lon remains clear, but on the surface a wrinkled membrane appears ; 
at the same time a disagreeable odor develops, and a marked indol 
reaction is then obtained. When injected as such the organism was 
not pathogenic for guinea-pigs, while inoculation together with ster- 
ile butter produced changes identical with those obtained by the 
same observer in the case of an acid-resisting bacillus which has 
repeatedly been found in butter. Unlike Pappenheim's organism, 
the bacillus which was isolated by Rabinowitch was not decolorized 
by Pappenheim's method. Nevertheless, she regards the two as 
identical, and looks upon similar acid-resisting bacilli which have 
been obtained from butter, manure, and various grasses, as closely 
related organisms. 

The Typhoid Bacillus. — It has been conclusively shown that the 
typhoid bacillus can be present in the sputum of typhoid patients, 
especially if there is a coexistent bronchitis or pneumonia. 1 

The plague bacillus is seen in the sputum in enormous numbers in 
cases of the pneumonic type of the disease. By direct observation, 
however, it may not be recognized immediately, and it is best in every 
case to resort to culture as well (see page 181.) The organism may 
be found in the sputum on the first day of the disease. 

Actinomycosis of the lungs may at times be diagnosed from the 
presence of the characteristic granules and thread-like formations in 
the sputum. Up to and including the 6 cases reported by Ewing 2 
in 1902, there are records of 100 American cases. 

The organism in question (Fig. 88) the streptoihrix actinomycotica 
or ray fungus probably belongs to the species cladothrix and occu- 
pies a unique position among the pathogenic bacteria. Infection in 
man and animals (cattle and pigs) possibly occurs through ears of 
barley or rye, a supposition, with which the observation accords that 
the disease frequently begins in the autumnal months. 

In the pus derived from ulcerating actinomycotic tumors, in the 
sputum in cases of pulmonary actinomycosis, and also in the feces 
when the disease has attacked the intestines, yellow granules 
will be observed, measuring from 0.5 to 2 mm. in diameter. If 
such a granule is examined microscopically, slight pressure being 
applied to the cover-glass, it will be seen to consist of numerous 

1 M. W. Richardsen, Boston Med. and Surg. Jour., Feb. 5, 1903. 

2 W. G. Ewing, Johns Hopkins Hosp. Bull., 1902, vol. xiii. J. Ruhrah, Annals of 
Surg., 1899, vol. xxx. (analysis of 62 cases). 



MICROSCOPICAL EXAMINATION. 



363 



threads which radiate from a centre in a fan-like manner and 
present club-shaped extremities. 

The organism may be demonstrated in the following manner : 
dried cover-glass preparations are stained for five to ten minutes 
with a saturated anilin-water and gentian-violet mixture (see page 
•146), when they are rinsed in normal salt-solution, dried between 
filter-paper, and transferred for two or three minutes to a solution 
of iodo-potassic iodide (1 : 100 or 1 : 50). They are then again 
dried between layers of filter-paper, decolorized in xylol-anilin oil 



Fig. 88. 





Actinomyces. (MrssER.) 

(1 : 2), washed in xylol, and mounted in balsam. The mycelium 
assumes a dark-blue color. 1 

Non-pathogenic Organisms. — Of the non-pathogenic micro- 
organisms which may be observed in sputa little is known. 

Oidium albicans may be seen in children, and is usually derived 
from the mouth. 

Of other fungi which are occasionally observed, there may be 
mentioned the Aspergillus fumigatus and Mucor corymbifer. Sac- 
charomyces has been seen in pus from pulmonary abscesses. Sar- 
cina pulmonalis has been found at times, and especially in the 
so-called mycotic bronchial props occurring in putrid bronchitis. 
They are usually smaller than the Sarcinae ventriculi, but larger 
than those observed in the urine ; they present the characteristic 
form of the latter. Various other bacilli and micrococci, in addi- 
tion to those mentioned, are also found in the sputa in large num- 
bers, but have not been closely studied, excepting the pus-organisms, 
which may almost always be demonstrated. 

Crystals. — Of crystals which may occur in sputa, it will be neces- 
sary to consider briefly the crystals of Charcot-Leyden, hseinatoidin, 



1 E. Paltauf, Sitzungsber. d. K. K. Gesellsch. d. Aerzte Wien, 1886. 



364 THE SPUTUM. 

cholesterin, margarin, tyrosin, calcium oxalate, and triple phos- 
phates. 

Charcot-Leyden Crystals. 1 — These crystals were discovered in the 
sputa of patients suffering from bronchial asthma, and were supposed 
to stand in a causative relation to the disease. This view, however, 
has been abandoned, and it is now known that they may occur in 
other diseases as well. But while their presence is almost constant 
in true bronchial asthma at a time when Curschmann's spirals can 
also be demonstrated in the sputa, they are only exceptionally met 
with in other diseases, such as acute and chronic bronchitis, phthisis, 
etc. They were formerly regarded as identical with Bbttcher's 
sperma crystals, but modern research has shown that this is not the 
case. They are straight hexagonal double pyramids, and appear 
under the microscope as flattened needles of variable size (Fig. 71). 
Some attain a length of from 40 fi to 60 //, while others are scarcely 
visible even with a comparatively high power of the microscope. 
They show a feeble, positive double refraction, and have but one 
optical axis, while the sperma crystals are biaxial and strongly 
double refracting. Their behavior to solvents is essentially the 
same as that of the sperma crystals, but they differ from these in 
their insolubility in formol. They are colored yellow with Florence's 
reagent, while the sperma crystals are stained a bluish black. Very 
curiously the appearance of Charcot-Leyden crystals is closely asso- 
ciated with the presence of eosinophilic leucocytes, and they have 
hence not inaptly been termed leucocytic crystals. They may in fact 
originate within the cells. In true bronchial asthma it is not un- 
common to find microscopical preparations of the sputum literally 
studded with eosinophilic leucocytes and free granules. Outside the 
sputum they are also found in the blood in myelogenous leukaemia, 
and in the stools in association with animal parasites. They readily 
form in both normal and abnormal red bone-marrow, and excellent 
specimens may be obtained for purposes of demonstration if a piece 
of a rib is allowed to remain exposed to the air for a few days. The 
marrow then usually contains large numbers. The crystals also form 
in decomposing viscera in general, and at times form a complete 
covering of old anatomical preparations. Their occurrence may 
be regarded as evidence of retrogressive changes in the cellular 
elements of any organ. Of the relation which they bear to the 
eosinophilic leucocytes, with which they are so constantly associated, 
nothing is known. The Charcot-Leyden crystals can be stained 
with the triacid stain, with thionin, with the eosinate of methylene- 
blue, and others. 

Hsematoidin crystals may be observed in the sputa following ex- 

1 Leyden. Virchow's Archiv, 1872, vol. liv. p. 324. Schreiner, Liebig's Annal., 
1878, vol. cxciv. p. 68. Colm, Centralbl. f. allg. Path. u. path. Anat., xol. x. p. 940. 
Brown, Phila. Med. Jour., 1898, p. 1076. 



THE PNEUMOCONIOSES. 365 

travasations of blood into the lung. They frequently occur in the 
form of ruby-red columns or needles ; amorphous granules, how- 
ever, are also seen, enclosed in the bodies of leucocytes, in which case 
they are probably always indicative of a previous hemorrhage, while 
the needles are generally observed when an abscess or empyema has 
perforated into the lungs. The substance is derived from blood- 
pigment, and is now known to be identical with bilirubin. 

Cholesterin crystals are at times seen in the sputa in cases of 
phthisis, pulmonary abscess, and, in general, whenever old accumula- 
tions of pus have entered the lung from a neighboring organ. They 
are readily recognized by their characteristic form and chemical 
properties (see Feces, page 282). 

Fatty acid crystals are frequently observed in cases of putrid bron- 
chitis and gangrene of the lung, and also in cases of bronchiectasis 
and phthisis. They occur in the form of single needles or groups 
of needles, which are long and pointed. They are easily soluble in 
ether and hot alcohol ; insoluble in water and acids. Chemically, 
they are probably composed of the higher fatty acids, such as pal- 
mitic and stearic acids. 

Tyrosin crystals have been observed in cases of putrid bronchitis, 
perforating empyema, etc. Leucin is likewise probably always pres- 
ent, occurring in the form of highly refractive globules. For the 
recognition of these bodies, particulary of tyrosin, a chemical exami- 
nation should always be made, as crystals of the soaps of fatty acids 
have frequently been mistaken for those of tyrosin (see Urine). 

Calcium oxalate crystals are rarely seen. Furbringer observed 
them in large numbers in a case of diabetes, and Unger found them 
in a case of asthma. They are readily recognized by their envelope- 
form, but they occur also in amorphous masses. They are soluble 
in mineral acids ; insoluble in water, alkalies, organic acids, alcohol, 
and ether. 

Triple phosphate crystals also are rarely seen, but may occur in 
cases of perforating abscesses, etc. They are recognized by their 
coffin-lid shape and the readiness with which they dissolve in acetic 
acid. 

THE PNEUMOCONIOSES. 

Anthracosis. — To some extent particles of carbon may be found 
in the sputum of almost every individual, and especially in smokers. 
The expectoration in such cases is of a pearl-gray color, and is 
brought up in larger or smaller masses, especially in the morning 
upon rising. Larger amounts are noted in miners and in those 
who are brought into close contact with coal-dust. Microscopically, 
particles of carbon and epithelial cells, especially of the alveolar 
type, as well as leucocytes loaded with the pigment, are seen. 

Siderosis. — In siderosis the sputum presents a brownish-black 



366 THE SPUTUM. 

color and contains cells enclosing particles of ferric oxide. These 
may be readily recognized by treating the preparation with a drop 
of ammoninni sulphide or potassium ferrocyanide solution in the 
presence of hydrochloric acid, when a black color on the one hand 
or a blue color on the other is obtained in the presence of iron. 
Chalicosis. — In chalicosis silicates are found in the sputa. 1 
Stycosis. — This condition was first described by A. Robin in a 
man, aged seventy, who from his seventeenth year suffered from 
cough and frequent attacks of diarrhoea, and whose condition at 
various times had been diagnosed as phthisis pulmonalis et intes- 
tinarum, although tubercle bacilli could not be demonstrated. The 
patient died from acute pericarditis complicating an attack of acute 
mono-articular rheumatism. Post mortem the lungs were found 
perfectly normal ; the bronchial and anterior mediastinal glands, as 
well as the mesenteric glands, however, were completely solidified 
and composed almost wholly of calcium sulphate. The man, it was 
then found, had been working in plaster of Paris all his life, and 
the symptoms observed — viz., cough, expectoration, and diarrhoea — 
Robin is inclined to attribute to the pressure of the solidified glands 
upon the bronchi and intestines. 

CHEMISTRY OF THE SPUTUM. 

In addition to the substances described, sputum contains certain 
albumins, volatile fatty acids, glycogen, ferments, and various inor- 
ganic salts. 

Among the albumins which have been observed in sputa may be 
mentioned serum -album in, and especially mucin, which is often pres- 
ent in large amounts. In pneumonic and purulent sputa albumoses 
also have been found. 

In order to demonstrate the presence of serum-albumin the sputa 
are treated with dilute acetic acid, when the filtrate is tested with 
potassium ferrocyanide, as described in the chapter on Urine. 
Serum-albumin is, of course, found in notable quantities in cases of 
oedema of the lungs. Especially interesting is the albuminous expec- 
toration which at times follows thoracentesis. The amount of sputum 
usually varies between 200 and 900 grammes, but may be much larger 
and may reach 2000 c.c. or even more. Occasionally it begins 
before the tapping is completed or immediately after. More com- 
monly, however, an interval varying from five minutes to one or 
two hours elapses before the expectoration begins. Its duration is 
variable. Sometimes it lasts only a few minutes, more often an hour 
or two, and in rarer cases a whole day or two. The condition is 
probably due to oedema of the lungs. 2 

1 Betts, Chalicosis Pulmonum," Jour. Am. Med. Assoc, 1900, No. 2. 

2 In the United States cases of albuminous expectoration following thoracentesis 
have been reported by Pepper, Allen, Pateck, and Eiesman. See especially the paper 
by Eiesman, in which a full account of the literature is given. Am. Jour. Med. Sci., 
April, 1902, p. 620. 



CHEMISTRY OF THE SPUTUM. 367 

The volatile fatty acids contained in sputa may be obtained by 
diluting with water, acidifying with phosphoric acid, and distilling, 
when the distillate is further examined as described in the chapter 
on Feces. Acetic, butyric, propionic, and capronic acid has been 
found. 

The fats and fixed fatty acids are extracted from the residue with 
ether, and shaken with a solution of sodium carbonate in order to 
transform them into their sodium salts, when the ether is decanted 
and evaporated, leaving the soaps behind. 

Glycogen has repeatedly been demonstrated in sputa, and may be 
detected by Ehrliclr's method (see page 138). 

The sputa of gangrene of the lung and putrid bronchitis have been 
shown to contain a ferment resembling trypsin. In order to test for 
this ferment, the sputa are extracted with glycerin ; the examination 
is then continued as described in the chapter on the Examination 
of Cystic Contents. 

The myelin granules, as I have already indicated, consist largely 
of protagon, lecithin, and cholesterin. 

The following are the inorganic salts which may be demonstrated 
in the sputum : sodium and magnesium chloride, phosphates of the 
alkalies and the alkaline earths (viz., calcium and magnesium), cal- 
cium and sodium sulphate and carbonate, phosphate of iron, and 
silicates. 



CHAPTEE VII. 

THE URINE. 

GENERAL CONSIDERATIONS. 

This is not the place to enter into a discussion of the various 
hypotheses which have been advanced to explain the manner in 
which waste-material is removed from the body through the kidneys. 
It will suffice to state that while the water and mineral constituents 
of the urine in part at least undoubtedly pass into the uriniferous 
tubules by a process of transudation, a selective glandular activity 
of the cells lining the convoluted tubules and the loop of Henle 
appears to be necessary for the elimination of the most important 
organic constituents. 

As the physical characteristics of the urine, as well as its chemi- 
cal composition, are influenced not only by the age and sex of the 
individual, but also by the character of the food ingested, the proc- 
ess of digestion, exercise, climate, temperature, race, etc., it is 
apparent that a quantitative analysis of any one urine, or even 
average figures, can give only an approximate idea of its composi- 
tion. The reader is accordingly referred for information to the 
special paragraphs concerning the variations in the individual con- 
stituents observed in health. It is important, however, to note 
that, notwithstanding the fairly wide variations here observed, the 
composition of the blood, as pointed out in a previous chapter, 
remains quite constant, showing the perfect manner in which the 
nervous system through the kidneys guards against an undue accu- 
mulation of what may be termed normal waste-products in the 
blood, and in virtue of which abnormal substances are also imme- 
diately eliminated. Moreover, as will be pointed out later on, a 
perfect mechanism appears to exist which prevents an undue accu- 
mulation of material in the blood that can hardly be regarded as 
waste. The presence of an amount of sugar in the blood exceeding 
6 pro mille, for example, appears to be invariably followed by gluco- 
suria, and the introduction of excessive quantities of sodium chloride 
similarly and almost immediately leads to an elimination of the 
excess. 



GENERAL CHARACTERISTICS OF THE URINE. 369 

GENERAL CHARACTERISTICS OF THE URINE. 
Appearance. 

Normal urine, just voided at an ordinary temperature, is either 
perfectly clear or but faintly cloudy, owing to the fact that the acid 
and normal salts present are all soluble in water. It may be stated, 
as a general rule, that whenever a urine freshly passed presents a 
distinct cloudiness some abnormality exists. 

When allowed to stand for a time a light cloud develops, 
which gradually settles to the bottom, constituting the so-called 
nubecula of the ancients. Examined under the microscope this is 
found to contain a few round, granular cells, somewhat larger than 
normal leucocytes, the so-called mucous corpuscles, and a few pave- 
ment-epithelial cells, derived from the bladder or genital organs. 
Chemically the nubecula probably consists of traces of mucus. 

When kept for twenty-four hours at an ordinary temperature 
crystals of uric acid are frequently observed in addition to the 
above elements, usually presenting the so-called whetstone-form. 
If, however, the temperature at which the urine is kept approaches 
the freezing-point, the entire volume becomes cloudy, owing to pre- 
cipitation of acid urates, as these are much less soluble in cold than 
in warm water ; on standing they gradually settle to the bottom of 
the vessel, and form what is known as a sediment, while the super- 
natant fluid again becomes clear. 

If kept still longer exposed to the air, at the temperature of the 
room, the entire volume of urine again becomes cloudy, owing to a 
diminution of its normal acidity, the result being a precipitation of 
ammonio-magnesium phosphate, calcium phosphate, and still later, 
when the urine has become alkaline, of ammonium urate. 

Gradually a heavy sediment, containing these salts in addition to 
the constituents of the primitive nubecula, forms at the bottom of 
the vessel ; the supernatant fluid, however, remains cloudy. On 
microscopical examination it will be seen that this cloudiness is due 
to the presence of enormous numbers of bacteria. 

The changes which take place in a normal urine when allowed 
to stand at ordinary temperature may be tabulated as follows : 

(1) Urine clear, no sediment — reaction acid. 

(2) Urine slightly cloudy, owing to development of the nubecula 

— reaction acid. 

^r , , f Mucous corpuscles, 
JNubecuia | Pavement-epithelial cells. 

(3) Urine clear, the nubecula has settled — reaction acid. 

f Mucous corpuscles, 

,. Epithelial cells, 

Sediment j Uric acid crystals, 

[ A few bacteria. 
24 



370 THE URINE. 

(4) Urine cloudy, owing to the precipitation of phosphates — 

reaction faintly acid. 

(5) Urine cloudy, owing to the presence of bacteria — reaction 



alkaline. 



Sediment 



Bacteria, 

Mucous corpuscles, 
Epithelial cells, 
Triple phosphates, 
Tri-calcium phosphate, 
Ammonium urate. 



Color. 

The color of normal urine may vary from a very light yellow to 
a brownish red, the particular shade depending essentially upon the 
specific gravity, becoming lighter with a diminishing, and darker 
with an increasing density. Pathologically the same rule holds 
good, except in diabetes, in which a very high specific gravity is gen- 
erally associated with a very light color. The reaction of the urine 
also exerts a marked influence upon its color, an acid urine being 
more highly colored than an alkaline urine, which can be readily 
demonstrated by allowing a specimen of acid urine to become alka- 
line, and by treating an alkaline urine with dilute hydrochloric or 
acetic acid. At the same time it may be said that every urine 
darkens slightly on standing, the reaction remaining acid. 

The various shades observed in normal urines may be grouped 
under the following headings : 

1. Pale urines vary from a faint yellow to a straw color. 

2. Normally colored urines are of a golden or an amber yellow. 

3. Highly colored urines present a reddish-yellow to a red color. 

4. Dark urines vary between brownish red and reddish brown. 
As these shades may occur in both normal and pathological urines, 

definite conclusions cannot, as a rule, be drawn from mere inspection. 
A very pale urine indicates simply an excess of water, which may 
be normal, but may also occur in such diseases as chronic interstitial 
nephritis, diabetes mellitus, diabetes insipidus, hysteria, and the 
various anaemias ; it is further seen during convalescence from acute 
febrile diseases, while a highly colored urine, though also occurring 
in health, may indicate the existence of a febrile process. It may 
be stated, *as a general rule, that a pale urine always excludes the 
existence of a febrile disease of any severity, and that the continued 
secretion of a very pale urine is usually associated with a certain 
degree of anaemia. 

The normal color of the urine is probably owing to the presence 
of several pigments, which are most likely closely related to each 
other and to hsematin. 

In addition to these colors others may be observed at times which 
are either pathological or accidental — i. e., due to the presence of cer- 



GENERAL CHARACTERISTICS OF THE URINE. 371 

tain drugs. The former are, on the whole, of greater importance to 
the physician than those mentioned above, as more definite conclu- 
sions can be drawn from their presence. Most important among 
such pathological pigments are those due : 

1. To the presence of blood-coloring matter. The color in such 
cases may vary from a bright carmin to a jet black, the exact shade 
depending upon the quantity of blood-coloring matter present, upon 
changes that the blood may have undergone either before or after 
being passed, and also upon the presence of the pigment in solution 
or contained in red corpuscles. 

2. Those due to the presence of biliary coloring matter. The 
color here varies from a greenish yellow to a greenish brown. 

3. A milky-colored urine is observed in cases of chyluria. 
Among the accidental abnormalities in color, on the other hand, 

are those due to the presence of substances like carbolic acid and its 
congeners, santonin, etc. 

As the recognition of the causes of such alterations, normal, 
pathological, and accidental, largely depends upon a more detailed 
study of the individual pigments, this subject will be dealt with 
more fully further on (see Pigments and Chromogens). 

Odor. 

The odor of the urine is usually of little significance. Normally 
it resembles that of bouillon, and in some cases that of oysters ; it 
is probably due to the presence of several volatile acids. The odor 
of urines undergoing decomposition is characteristic and has been 
termed " the urinous odor of urine," an ill-chosen term, as this odor 
is always indicative of an abnormal condition. 

The ingestion of asparagus, onions, oil of turpentine, etc., pro- 
duces a characteristic odor which is of no significance. 

Consistence. 

Urine, Avhile normally fluid and but slightly viscid, may in dis- 
ease acquire a marked degree of viscidity, which becomes especially 
apparent upon attempting its filtration ; the liquid passes through 
the paper with more and more difficulty, and finally clogs its 
pores altogether. 

Quantity. 

The quantity of the urine is normally subject to great variations, 
the amount eliminated in the twenty-four hours being influenced by 
that of the fluid ingested, the nature and quantity of the food, the 
process of digestion, the blood -pressure, the surrounding tempera- 
ture, sleep, exercise, body-weight, sex, age, etc. 

It is easy to understand, then, why figures given by different 



372 THE URINE. 

observers in different countries should vary considerably. Salkow- 
ski, in Germany, thus gives 1500 to 1700 c.c. as the normal 
amount; v. Jaksch, in Austria, 1500 to 2000 c.c. ; Landois and 
Sterling, in England, 1000 to 1500 c.c. ; Gautier, in France, 1250 
to 1300 c.c. In the United States I have found an average secre- 
tion of from 1000 to 1200 c.c. in the adult male, and 900 to 
1000 c.c. in the adult female. It is thus seen that the secretion 
of urine is greatest in Germany and Austria, where the body -weight 
and ingestion of liquids are greater than in England, France, and 
the United States. 

Children pass less, but relatively more (considering their body- 
weight) urine than adults. 

Women pass somewhat less than men. 

During the summer months, when a larger proportion of water 
is eliminated through the skin and lungs than in cold weather, less 
urine is voided. The same occurs during repose, more urine being 
passed during active exercise, and hence less during the night than 
during the day. 

The amount of urine secreted in the different hours of the day 
varies greatly, reaching its maximum a few hours after meals. It 
decreases toward night, and reaches its lowest point in the first hours 
of the night, after which it begins to rise rapidly until 2 or 3 
o'clock in the morning. 

The ingestion of large amounts of liquid, of course, increases the 
daily amount considerably, and 3000 c.c. may be passed under such 
conditions by an individual in good health, w T hile it may decrease to 
800 or 900 c.c. when but little liquid is taken. 

After the ingestion of much solid food the secretion of urine is 
temporarily diminished. 

Water containing no salts possesses distinctly diuretic properties, 
as do also beer, wine, coffee, tea, etc. 

The most important medicinal diuretics are digitalis, squill, 
broom, spirit of nitrous ether, juniper, urea, etc. 

Pathologically the amount of urine varies within wide limits. In 
a given case, moreover, it may be exceedingly difficult to determine 
whether or not the secretion is within physiological limits. As a 
general rule, whenever less than 500 c.c. or more than 3000 c.c. are 
passed some abnormal condition exists, providing all other causes 
which might lead to the secretion of such an amount can be elimi- 
nated. 

Clinically we speak of polyuria and oliguria. 

Polyuria. — Polyuria is observed in many diseases, and is present 
under such varied conditions that a classification is only warrant- 
able upon a hypothetical basis, especially as the causative factors 
concerned in its production are mostly unknown. 

As polyuria is almost invariably associated with diabetes mel- 



GENERAL CHARACTERISTICS OF THE URINE. , 373 

litus, its presence in any case should always excite suspicion and 
lead to a proper examination. The quantity of fluid eliminated in 
diabetes is usually dependent upon the amount ingested. The excre- 
tion of a proportionately large amount of fluid, however, does not 
necessarily follow the ingestion directly, and retention of a large 
amount may occur ; it has been shown, as a matter of fact, that the 
diabetic patient excretes liquids with greater difficulty than the 
healthy subject. At the same time it should be borne in mind that 
the polyuria in diabetes is not necessarily continuous, and that 
periods during which a normal or even a subnormal amount of urine 
is observed may alternate with true polyuria. From 2 to 26 or even 
50 liters may be passed within twenty-four hours. Intercurrent dis- 
eases of a febrile character may modify the quantity very materially 
and cause the elimination of a normal or subnormal amount. 

The cause of the polyuria in diabetes mellitus is unknown. The 
ingestion of large amounts of liquids, of course, leads to a cor- 
respondingly large elimination, and the existing polydipsia could, 
hence, be made responsible for the polyuria ; the latter would thus 
be the result of an increased stimulation of the thirst-centre, pos- 
sibly owing to the presence of some abnormal constituent of the 
blood. The polydipsia, however, may also be the result of a pri- 
mary polyuria. 

The polyuria associated with the resorption of large pericardial, 
pleural, ascitic, and subcutaneous effusions is more readily under- 
stood, although the primum mobile may be unknown ; it depends 
in such cases entirely upon the presence of excessive quantities of 
fluid in the bloodvessels. 

A form of polyuria which has been termed " epicritic polyuria " 
is frequently observed during convalescence from acute febrile dis- 
eases, and is of prognostic importance. Its occurrence in a given 
case is regarded by many as a good omen, especially in typhoid 
fever ; still it must not be forgotten that a polyuria may occur 
after subsidence of the fever, and be followed by a considerable 
degree of oliguria, and in some cases may precede death. A polyuria 
of this kind probably always indicates the elimination of waste- 
products which had accumulated in the blood during the course of 
the disease, but it may, at the same time, be due to the presence of 
retained water. 

Second in constancy is the polyuria associated with granular 
atrophy of the kidneys, constituting one of the most important symp- 
toms of the disease. Cases have been reported in which as much as 
10,000 c.c. of urine were secreted in the twenty-four hours ; 2000 
to 4000 c.c. represent the usual amount in such cases. Polydipsia 
commonly exists at the same time, and the explanation of the poly- 
uria again becomes a very difficult matter. That generally given 
is based upon the following considerations : 



374 THE URINE, 

In granular atrophy of the kidneys large tracts of renal paren- 
chyma are destroyed, the result being a diminution in the area of 
glandular material, which in itself would lead to a diminished secre- 
tion of urine. The coexisting cardiac hypertrophy, however, by 
raising the blood-pressure in the kidneys, is supposed to counter- 
balance the renal deficiency and even to lead to an increase in the 
amount of urine. There appears to be some doubt as to the cor- 
rectness of such an explanation, however, as the existence of hyper- 
trophy of the left ventricle in the absence of glandular disease of 
the kidneys by no means leads to a degree of polyuria comparable 
to that observed in this disease. It is possible that while cardiac 
hypertrophy in itself may be one of the causative factors, still 
another may be a vicarious action of the sound glandular elements. 
If such be the correct explanation, the coexisting polydipsia is 
merely secondary. This, however, can only be regarded as an 
hypothesis, and the diminished renal secretion associated with a 
gradually developing cardiac dilatation should not be upheld as 
an absolute proof of its correctness. 

Very curiously, polyuria may occur also in association with mul- 
tiple myelomata of the bones and the presence of Bence Jones 7 
albumin in the urine. In one of the cases reported by Hamburger, 1 
and which I had occasion to study in greater detail from a chemical 
point of view, 3500 c.c. were voided in the twenty-four hours. 
The symptom, however, is not constant. 

Polyuria, furthermore, has been observed in the most diverse 
diseases of the nervous system, both functional and organic. It is 
frequently observed both as a transitory and a permanent symptom 
in cases of hysteria. Large quantities of a very pale urine are 
secreted after the occurrence of severe hysterical seizures, but the 
same may be observed throughout the course of the disease. A 
similar condition is frequently seen in neurasthenia, migraine, chorea, 
and epilepsy. 

Generally speaking, it may be said that a paroxysmal polyuria in 
nervous diseases is associated with functional derangement, while 
a continuous polyuria appears to be connected rather with true 
organic changes. It has been observed in certain cases of tabes, 
cerebrospinal and spinal meningitis, during the first stage of general 
paresis, in association with tumors involving the medulla, the cere- 
bellum, and the spinal cord, in injuries affecting the central nervous 
system, in Basedow's disease, etc. Cases of idiopathic diabetes 
insipidus also should probably be classified under this heading. 
Enormous quantities of urine may be secreted in this disease, which 
are equalled only by cases of diabetes mellitus, and may at times 
reach 43 liters per diem. 

1 L. P. Hamburger, " Two Examples of Bence Jones Albuminosuria associated with 
Multiple Myeloma," Johns Hopkins Hosp. Bull., Feb., 1901. 



GENERAL CHARACTERISTICS OF THE URINE. 375 

Oliguria. — Oliguria is, on the whole, more frequent than polyuria, 
and is met with in almost all conditions associated with a lowered 
blood-pressure. First in order stand those cases of cardiac disease 
in which compensation has failed, whether the cardiac weakness is 
primary or occurring secondarily to other diseases — i. e., pulmonary, 
hepatic, and renal. 

The oliguria observed in the so-called continued fevers, notably 
typhoid fever, is probably also referable to cardiac weakness. It 
should be remembered, however, that a larger proportion of water 
is eliminated ^through the skin and lungs than normally, and that 
a retention of fluids also undoubtedly occurs which is not due to 
cardiac weakness ; still other factors may be concerned in its 
production. 

The oliguria occurring in acute nephritis and in chronic paren- 
chymatous nephritis in all probability depends largely upon mechani- 
cal causes, the increased intra-canalicular resistance in the form of 
desquamated epithelium and tube-casts, as well as the pressure of 
the exudate upon the bloodvessels obstructing the passage of urine, 
while the functional activity of the diseased glandular elements is at 
the same time lowered. Upon mechanical causes, also, depend all 
those cases of oliguria which are associated with the presence of a 
stone or tumor pressing upon a portion of the urinary tract. 

Oliguria may occur as a nervous manifestation in connection with 
puerperal eclampsia, lead colic, hysteria, psychic depression, preced- 
ing and during epileptic seizures, etc. Whenever there is a diminu- 
tion in the amount of bodily fluids oliguria is also observed ; this is 
particularly marked in cholera and following severe hemorrhage. 

Obstruction to the flow of blood in the vena cava or liver, lead- 
ing to an increase of venous pressure and a decrease of arterial 
pressure in the kidneys, likewise results in oliguria, as is seen in 
atrophic hepatic cirrhosis, acute yellow atrophy, thrombosis of the 
vena cava and the renal vein, or in cases in which pressure is 
exerted upon these by tumors, ascitic fluid, etc. 

In any case the oliguria may go on to complete anuria, which 
condition not infrequently precedes death. Anuria may, however, 
also occur independently of a pre-existing oliguria, as in hysteria. 

Specific Gravity. 

The specific gravity of normal urine varies between 1.015 and 
1.025, corresponding to 1200 to 1500 c.c, viz., the normal amount 
of urine voided in twenty-four hours. Pathologically, a specific 
gravity of 1.002 on the one hand and 1.060 on the other may occur, 
depending upon the amount of solids and fluids present, increasing 
as the solids increase, the amount of urine remaining the same, and 
decreasing as the amount of fluid increases, the solids remaining the 



376 THE URINE. 

same. The specific gravity is thus an index in a general way of 
the metabolic processes taking place in the body. 

The necessity of determining the specific gravity of the total 
amount of urine voided in a given case, and not that of an individual 
specimen passed during the twenty-four hours, becomes apparent 
upon considering the variations which may occur in the quantity of 
solids and liquids ingested during the day. The ingestion of large 
amounts of water or beer would, of course, result in the passage of 
a correspondingly large quantity of urine within the next few hours, 
containing but a small amount of solids, and hence presenting a low 
specific gravity. From such an observation it would be erroneous 
to infer a diminished excretion of solids for the day, as succeeding 
specimens would in all probability be passed presenting a higher 
specific gravity. An observation made upon a specimen taken 
from the collected urine of the twenty-four hours, moreover, can 
only then convey a correct idea if the total quantity is within the 
normal limits. If this should not be the case, the volume of the 
urine passed must first be reduced to the normal and the specific 
gravity then taken. 

Supposing a known quantity of common salt to be dissolved in 
1000 c.c. of water, so that the resulting specific gravity is 1.24 ; by 
doubling the amount of salt and water the specific gravity would 
still remain the same, while the amount of salt would actually be 
twice as large as at first. In order to obtain the specific gravity 
indicating the actual amount of solids present it would be necessary 
to concentrate the fluid to 1000 c.c. The specific gravity being 
inversely proportionate to the amount of fluid secreted, the necessary 
correction is made according to the following formula : 

Sp.g,= $ 

in which Sp. gr. indicates the specific gravity to be determined, q 
the amount of urine actually passed, d the specific gravity observed, 
and N the normal amount of urine — L <?., 1200 c.c. 

Example. — A patient has passed 3000 c.c. of urine in the twenty- 
four hours with a specific gravity of 1.017 ; this is corrected accord- 
ing to the above formula : 

Sp.gr. =3000_XJZ = i042i 

F s 1200 

From the specific gravity the amount of solids can be calculated 
with sufficient accuracy for clinical purposes by multiplying the last 
two decimal points by 2, the number obtained indicating the amount 
of solids in 1000 c.c. of urine. 

To illustrate the necessity of either indicating the total amount of 
urine passed within the twenty-four hours, and of taking the specific 



GENERAL CHARACTERISTICS OF THE URINE. 377 

gravity from this collected urine, or of correcting the specific gravity 
as shown above, the following case may be supposed : 

A " specimen " of urine is taken, presenting a specific gravity of 
1.002 ; by multiplying the 2 by 2, the person would be supposed to 
pass 4 grammes of solids in every 1000 c.c. of urine. Had the 
specific gravity been observed in the total amount of urine passed 
in the same twenty-four hours, it would have been found to be 1.012, 
the man having passed 3000 c.c. of urine; by multiplying 12 by 2, 
24 grammes of solids would have represented the amount in every 
1000 c.c. — ire., 24 X 3 = 72 grammes in toto. The same result 
would have been reached by correcting the specific gravity of 1.012 
for the normal amount of urine. 

The first calculation then would have indicated a considerable 
deficit as compared with the second. 

The following rules for practice may thus be stated : 

1. Whenever the specific gravity only is to be indicated in a uri- 
nary report it should always be the corrected one ; if this is not done, 
the amount of urine should be stated in every case. 

2. The specific gravity should always be taken from a specimen 
of the collected urine of the twenty-four hours, and never from a 
specimen ad libitum. 

From the rule, that the specific gravity of a urine is inversely 
proportionate to the amount of fluid eliminated, it must follow that 
whatever causes produce oliguria will also produce a high specific 
gravity, while all those causes which produce a polyuria will similarly 
produce a low specific gravity, with the following exceptions : 

1. A diminished amount of urine with a lowered specific gravity 
occurs in many chronic diseases and toward the fatal termination of 
acute diseases, indicating a defective elimination of solids. 

2. The same may be observed in certain cases of cedema. 

3. Following copious diarrhoea, vomiting, and sweating. 

4. A high specific gravity is associated with polyuria in diabetes 
mellitus. 

Unfortunately the determination of the specific gravity and the 
solids contained in urines does not furnish as valuable information 
in many cases as would be expected d priori. This is largely owing 
to the fact that the organic constituents of the urine have a lower 
specific gravity than the inorganic salts, and especially the chlorides, 
which are usually present in considerable amount. It thus not in- 
frequently happens that the nitrogenous constituents are considerably 
increased, while the specific gravity is relatively low, owing to the 
absence or a diminution in the amount of chlorides. In other words, 
while the specific gravity may be regarded as a fair index of the 
total amount of solids excreted, its increase or decrease furnishes no 
information as to the nature of the constituents causing such a 
change. 



378 



THE URINE. 



Fig 



Determination of the Specific Gravity. — The specific gravity of 
the urine is most conveniently determined by means of a hydrometer 
indicating degrees varying from 1.002 to 1.040. Such instruments, 

constructed especially for the examina- 
tion of urine, are termed urinometers 
(Fig. 89). A good instrument should 
have a stem upon which the individual 
divisions are at least 1.5 mm. apart, and 
each division should correspond to 0.5 
degree. 

Urinometers may also be purchased 

which are provided with a thermometer, 

a matter of great convenience. Every 

instrument should be carefully tested by 

fjl (I- comparison with a standard hydrom- 

1 1 1 1^ eter * 

In Order to determine the specific 

gravity in a given case a cylindrical ves- 
sel is nearly filled with urine and the 
urinometer slowly introduced, the read- 
ing being taken at the lower meniscus 
as soon as the instrument has come to 
rest. 

Precautions : 1. The urinometer must 
be given ample room, and the read- 
ing should never be taken when the in- 
strument touches the sides of the ves- 
sel, as owing to capillary attraction it 
is otherwise raised, causing the reading 
to be too high. 

2. The instrument must be perfectly 
dry and clean before being used, and 
should never be allowed to "drop" into 
the urine, as otherwise the weight of the 
instrument is increased by adhering 
drops of water, and the reading is too low. 

3. Any foam upon the surface of the urine should first be removed 
by means of a piece of filter-paper, as it interferes with the accuracy 
of the reading; bubbles of air adhering to the instrument, and 
thereby elevating it, should be carefully removed with a feather. 

4. The specific gravity should always be determined in specimens 
taken from the twenty-four-hour urine, and corrected according to 
the formula given above. 

5. If the quantity of urine is too small to determine its specific 
gravity with a urinometer, the following method may be advan- 
tageously employed : 




Urinometer. (W. Simon.) 



GENERAL CHARACTERISTICS OF THE URINE. 



379 



About 50 c.c. of urine are measured into a small bottle pro- 
vided with a ground-glass stopper, or into a pyknometer like the one 
pictured in Fig. 90, and accurately weighed. The weight of the 

Fig. 90. 




The pyknometer. 

urine divided by its volume gives the specific gravity, which must, 
however, be corrected for the temperature of the urine. If accuracy 
is required, such corrections should be made in every case, as the 
specific gravity increases or decreases by one degree for every three 
degrees C. above or below the point for which the instrument is reg- 
istered, viz., 15° C. According to Bouchardat and Mercier, this 
method is not strictly accurate, and the following table has been 
constructed by which the proper corrections can be readily made : 



Tempera- 


Normal 


Sugar 


Tempera- 


Normal 


Sugar 


ture. 


urine. 


urine. 


ture. 


urine. 


urine. 


0° 


0.9 


1.3 


18° 


0.3 


0.6 


1 


0.9 


1.3 


19 


0.5 


0.8 


2 


0.9 


1.3 


20 


0.9 


1.0 


3 


0.9 


1.3 


21 


0.9 


1.2 


4 


0.9 


1.3 


22 


1.1 


1.4 


5 


0.9 


1.3 


23 


1.3 


1.6 


6 


0.8 


1.2 


24 


1.5 


1.9 


7 


0.8 


1.1 


25 


1.7 


2.2 


8 


0.7 


1.0 


26 


2.0 


2.5 


9 


0.6 


0.9 


27 


2.3 


2.8 


10 


0.5 


0.8 


28 


2.5 


3.1 


11 


0.4 


0.7 


29 


2.7 


3.4 


12 


0.3 


0.6 


30 


3.0 


3.7 


13 


0.2 


0.4 


31 


3.3 


4.0 


14 


0.1 


0.2 


32 


3.6 


4.3 


15 . 


0.0 


0.0 


33 


3.9 


4.7 


16 


0.1 


0.2 


34 


4.2 


5.1 


17 


0.2 


0.4 


35 


4.6 


5.5 



Example. — Supposing the specific gravity to have been 1.030, at 
a temperature of 20° C, it would be necessary to add 0.9 to 1.030, 



380 



THE URINE. 



making this 1.0309 ; at a temperature of 10° C, it would similarly 
be necessary to subtract 0.5. 

Determination of the Solid Constituents. — As indicated above, 
the amount of solids can be calculated with a degree of accuracy 
sufficient for clinical purposes by multiplying the last two figures of 
the specific gravity by 2 ; the number obtained indicates the amount 
of solids in every 1000 c.c. of urine. If greater accuracy is re- 
quired, the following method may be employed : 

Five c.c. of urine, accurately measured, are placed in a watch- 
crystal containing a little dry sand (sand and crystal having been 
previously weighed) ; this is placed over a dish containing concen- 
trated sulphuric acid, and under the receiver of an air pump which 
has been made perfectly air-tight by thoroughly lubricating the 
ground-glass edge of the bell with mutton tallow and applying the 
bell with a slightly grinding movement to the ground-glass plate. 
The receiver is now exhausted and the urine allowed to remain in 
the vacuum for twenty-four hours, when the bell is again exhausted 
and left for twenty -four hours longer ; at the end of this time the 
crystal is weighed, the difference between the two weights obtained 
indicating the amount of solids in 5 c.c. of urine, from which the 
percentage and total amount are readily calculated. 

The slight loss of ammonia which results when this method is 
employed scarcely affects the accuracy of the result. 

REACTION. 

The reaction of the twenty-four-hour urine is, as a rule, acid ; 
individual specimens, passed in the course of the same twenty-four 
hours, may be either alkaline, acid, or amphoteric. 

When a mixture of different acids is brought into contact with 
a mixture of alkalies, the acids combine with the alkalies according 
to the degree of affinity which exists between them and the amount 
present of each. Upon the excess of acids over alkalies, and vice 
versa, depends the resulting reaction. If the alkalies are not suf- 
ficient in amount to saturate the acids, an acid reaction will result, 
while an insufficient amount of acid will give rise to an alkaline 
reaction. The same principle holds good for the acids and alka- 
lies giving rise to the salts present in the urine. As here the 
alkaline substances are not present in sufficient amount to saturate 
the acids, which can readily be seen from the following table, the 
acid reaction of normal urine is explained : 



HC1 


so 3 


PA 


K 


Na 


NH 3 


Ca- 


Mg 


10.1265 
6.3811 


2.3157 
1.3315 


3.0334 

0.9827 


2.5830 
1.5194 


5.4780 
5.4780 


0.5977 

0.8087 


0.0405 
0.0233 


0.0880 
0.0843 



The figures in the first column indicate the average daily amount 



REACTION. 381 

of the inorganic acids and alkalies present in the urine of twenty- 
four hours, and the figures in the second column their equivalents 
in terms of sodium, that of phosphoric acid having been estimated 
as diacid sodium phosphate. From this it is seen that the acid 
equivalents, 8.6953, exceed the alkaline equivalents, 7.9137, by 
0.7816 gramme of sodium. There are present then in the urine, in 
addition to the normal salts of the monobasic acids, acid salts and 
especially diacid sodium phosphate, ]SaH 2 P0 4 . To the latter the 
acidity of the urine is due. If, on the other hand, the alkalies 
exceed the acids in amount, an alkaline urine will result, which may 
occur physiologically under various conditions. 1 

The so-called amphoteric reaction will be observed when the 
diacid and neutral sodium phosphates, NaH 2 P0 4 and Na 2 HP0 4 , are 
present in a certain definite proportion ; the urine then changes the 
color of red litmus paper to blue, and vice versa. 2 

A neutral urine is never observed under normal conditions. The 
presence of a free acid, moreover, is not possible, as it would imme- 
diately combine with ammonia, which is constantly being set free in 
the tissues of the body as ammonium lactate, and is normally trans- 
formed into urea. 3 

The question now arises, How does the acidity of the urine re- 
sult ? and What are the ultimate factors that will produce an alka- 
line and an amphoteric reaction ? 

These are problems which as yet await a final answer. Our 
present ideas, however, may be formulated as follows : In the me- 
tabolism of the body -tissues acids are constantly produced ; chief 
among these is sulphuric acid, which results from albuminous decom- 
position, and hydrochloric acid, which at a certain period of digestion 
is reabsorbed from the stomach. As the alkalinity of the blood is 
due to neutral sodium phosphate and sodium carbonate, these salts 
are attacked by the free acids as soon as they enter the blood, the 
result being the formation of acid salts, and, as the latter diffuse 
more readily through an animal membrane than alkaline salts, the 
secretion of an acid urine from the alkaline blood is in part ex- 
plained. Nevertheless it is impossible to exclude a certain specific 
action on the part of the glandular elements of the kidneys, as 
otherwise the secretion of all glands, supposing this to depend 
upon a process of filtration or diffusion only, would necessarily be 
acid. 

As the alkalinity of the blood increases the acidity of the urine 
decreases, until finally an alkaline urine results. The degree of the 
alkalinity of the blood, however, depends essentially upon the nature 
of the food and the secretion of the gastric juice, viz., the hydro- 

^riicke, Maly's Jahresber., 1887. vol. xvii. p. 189. Liebig, Annal. d. Chem. u. 
Pharmakol., 1844, vol. 1. p. 61. 

2 Heintz, Jour. f. prakt. Chem., 1872, vol. vi. p. 274. 

3 F. Walters, Arch. f. exper. Path. u. Pharmakol., 1877, vol. vii. p. 148. 



382 THE URINE. 

chloric acid. The ingestion of vegetable food, rich In salts of or- 
ganic acids, which become oxidized in the body to the carbonates of 
the alkalies, will result in the passage of an alkaline urine, for the 
alkalies thus formed when absorbed into the blood are more than 
sufficient to neutralize completely all the acids present, and the elimi- 
nation of neutral sodium phosphate alone takes place. In the case 
of animal food the reverse holds good. The alkaline carbonates 
here formed are not sufficient to neutralize the excess of acids, and 
diacid phosphate of sodium is hence eliminated in large quantity. 1 

An amphoteric urine results whenever the elimination of neutral 
and acid sodium phosphate is the same ; such an occurrence is, 
therefore, more or less accidental. 

As the alkalinity of the blood is increased during the secretion 
of the acid gastric juice, it may frequently happen, especially follow- 
ing the ingestion of a large amount of food, that an alkaline urine 
is voided. If this does not take place, the acidity of the urine is at 
least diminished, but increases again during the process of resorp- 
tion of hydrochloric acid and peptones. The statement so generally 
found in text-books, that the urine secreted after a meal is alka- 
line, is not strictly correct ; in a series of observations which I made 
in this direction an alkaline urine was observed in only 20 per 
cent, of the cases examined. 2 

It may thus be stated that an alkaline urine will result under 
physiological conditions whenever the alkaline salts present in the 
food are sufficient to neutralize all the acids formed, as occurs in the 
case of a vegetable diet, and, furthermore, whenever the period of 
gastric secretion is lengthened. 

If an acid urine is allowed to stand exposed to the air for a cer- 
tain length of time, its degree of acidity gradually diminishes, and 
the reaction finally becomes alkaline. At the same time the urine 
becomes cloudy and deposits a sediment, which consists of ammonio- 
magnesium phosphate, MgNH 4 P0 4 +6H 2 0, neutral calcium phos- 
phate, Ca 3 (P0 4 ) 2 , and still later contains ammonium urate, C 5 H 2 - 
(NH 4 ) 2 N 4 O s , in addition to the constituents of the primitive nubecula 
— i. e., a few mucous corpuscles and pavement epithelial cells. The 
entire volume of urine, moreover, remains cloudy, owing to the 
presence of innumerable bacteria. The odor becomes extremely disa- 
greeable, and distinctly " urinous." In short, " ammoniacal decom- 
position " has occurred. This has been shown to depend upon the 
action of certain bacteria, notably the Micrococcus urea? and the Bac- 
terium urese, which are present in the air. 3 These organisms cause 
decomposition of the urea found in every urine, with the forma- 
tion of ammonium carbonate, according to the following equations : 

1 E. Salkowski u. J. Munk, Virchow's Archiv, 1877, vol. Ixxvi. p. 500. 

2 Quincke, Zeit. f. klin. Med., vol. vii. 

3 W. Leube, "Ueber die ammoniakalisehe Harngahrung," Virchow's Archiv, 1885, 
vol. c. p. 555. 



REACTION. 383 

CO(NH 2 ) 2 + 2H 2 = (NH 4 ) 2 C0 3 
(NH 4 ) 2 C0 3 = 2NH 3 + H 2 + C0 2 . 

It is not the bacterium, however, which directly produces the re- 
sult, but a bacterial product, and in this case an enzyme. 

An alkaline urine, the alkalinity of which is not due to ammo- 
niacal fermentation, however, but to other causes, as indicated 
above, may, of course, undergo the same change as an acid urine ; 
but it is necessary to distinguish sharply between these two varieties 
of alkaline urines, as the recognition of the cause of* the alkalinity is 
very often most important in diagnosis. The distinction is readily 
made by fastening a piece of sensitive red litmus-paper in the cork 
of the bottle containing the urine. If the alkalinity of the urine is 
due to the presence of ammonia, the litmus-paper will turn blue, but 
soon changes to red when exposed to the air ; while a urine, the 
alkalinity of which is due to the presence of fixed alkalies, will turn 
red litmus-paper blue only when immersed in the urine, the change 
in color at the same time persisting. 

As ammoniacal decomposition can also occur within the urinary 
passages, it is important, whenever an alkaline reaction due to the 
presence of ammonia is observed, to test the urine at once upon being 
voided, or, still better, to procure a portion with a catheter. Such 
urines are frequently seen in cases of cystitis the result of paralysis, 
urethral stricture, gonorrhoea, etc. In this connection it is interest- 
ing to note that whereas in old, neglected cases of cystitis an alkaline 
reaction is frequently observed, Brown has shown that in the great 
majority of cases of cystitis, both acute and chronic, and also in 
those of pyelitis and pyelonephritis, the urine is acid. 1 

An intensely acid reaction is observed in almost all concentrated 
urines, especially in fevers, in certain diseases of the stomach asso- 
ciated with a diminished or suspended secretion of hydrochloric acid, 
in gout, lithiasis, acute articular rheumatism, chronic Bright 7 s dis- 
ease, diabetes, leukaemia, scurvy, etc. Whenever a very acid urine 
is secreted for a considerable length of time, the possibility of renal 
irritation and the formation of concretions should be borne in mind. 

An alkaline urine, the alkalinity of which is not owing to the 
presence of ammonia, but to a fixed alkali, is observed in certain 
cases of debility, especially in the various forms of anaemia, follow- 
ing the resorption of alkaline transudates, the transfusion of blood, 
frequent vomiting, a prolonged cold bath, etc. It may also be due 
to the ingestion of certain drugs, viz., salts of the organic acids and 
alkaline carbonates, the former being transformed into the latter, as 
has been mentioned. An increase in the degree of acidity may 
similarly take place after the ingestion of mineral acids. 

Of interest is the observation of Pick 2 that in twenty-four to 

1 T. R. Brown, Johns Hopkins Hosp. Eep., 1901, vol. x. p. 11. 

2 F. Pick, "The Urine in Pneumonia," Munch, med. Woch., 1898, No. 17. 



384 THE URINE. 

forty-eight hours after the crisis in pneumonia the urine shows a 
marked decrease in its acidity, becoming neutral or even alkaline. 
This phenomenon, which was observed in thirty-one out of thirty- 
eight cases, persists for a day or a day and a half, and then the 
acidity returns. In all likelihood the change is due to absorption 
of the large amounts of sodium which are present in the exudate. 

An increase in the acidity of the urine upon standing has repeat- 
edly been observed, and is probably due to the formation of new 
acids from pre-existing acid-yielding substances, such as certain 
carbohydrates, alcohol, etc., which have undergone fermentation. 
This phenomenon is frequently observed in diabetic patients. 

A decrease in the acidity of normal urine upon standing, however, 
is the rule, owing to a gradual decomposition of sodium urate by 
the acid sodium phosphate, acid sodium urate, and, later on, uric 
acid resulting, which are thrown down as a sediment in consequence 
of the diminished acidity of the urine, and which, hence, no longer 
influence its reaction. This is shown in the equations : 

(1) NaH 2 P0 4 + C 5 H 2 Na 2 N 4 3 = Na 2 HP0 4 + C 5 H 3 NaN 4 3 

(2) NaH 2 P0 4 + C 5 H 3 NaN 4 3 = Na 2 HP0 4 + C 5 H 4 N 4 3 . 

Determination of the Acidity of the Urine. — The old method 
of titrating a certain amount of urine with a decinormal solution of 
sodium hydrate has been abandoned and replaced by that of Freund. 
This is essentially based upon the observation that the acid reaction 
of the urine is referable exclusively to diacid phosphates. 

Freund's Method. 1 — In 50 c.c. of urine the total amount of phos- 
phoric acid is estimated as described on page 331. The result is 
termed T. In a second portion of 50 c.c. the monacid phosphates, 
M, are then precipitated with a normal solution of barium chloride — 
i.e., one containing 122 grammes of the crystallized salt in 1000 
c.c. of water — 10 c.c. being added for every 100 mgrms. of the total 
amount of phosphoric acid found. After the addition of the barium 
the mixture is diluted to 100 c.c, filtered, and the phosphoric acid 
estimated in 50 c.c. of the filtrate. The result obtained is termed 
D. Owing to the fact that not only are the monophosphates pre- 
cipitated on the addition of the barium chloride, but also a small 
amount of normal phosphates, and that a small amount of diacid 
phosphate is formed at the same time and passes into solution, an 
error is incurred. This, however, remains constant, and amounts to 
3 per cent, in favor of the diacid phosphates. 

As the total amount of phosphoric acid is subject to fairly wide 
variations, even in health, it is best to calculate the relative propor- 
tion of T to D for 100 c.c. of urine, and then to determine the abso- 
lute degree of acidity for the twenty-four hours. Figures are thus 
obtained which are directly comparable with one another. 

1 E. Freund, Centralbl. f. d. med. Wiss., 1892, p. 689. 



CHEMISTRY OF THE URINE. 385 

Example. — Supposing that T amounted to 0.386 gramme for 100 
c.c. of urine, and D to 0.338 gramme. Three per cent, of D would 
have to be deducted for reasons just given, making the true value 
of D 0.3279. The relative proportion of T to D would then be 
84.9, as determined according to the equation 

0.386 : 0.3279 : : 100 : x ; and x = 84.9. 

Supposing, further, that the total amount of urine was 2000 c.c, 
the total acidity of the twenty-four hours would correspond to 1698, 
according to the equation 100 : 84.9 : : 2000 :x; and x = 1698, and 

the total acidity per hour to , i. e. y 70.7. 

The results obtained can also be expressed in terms of hydrochloric 
acid, 100 mgrms. of the diacid phosphates corresponding to 102.8 
mgrms. of hydrochloric acid. This mode of indicating the total 
acidity of the urine would actually be the better. 

If the urine should be alkaline and cloudy, the sediment is first 
dissolved by carefully adding a one-tenth or one-fourth normal solu- 
tion of hydrochloric acid, the amount added being then deducted 
from the total acidity. Should negative values be found, these could 
be expressed in terms of sodium hydrate. 1 

With this method a complete revision of all the work previously 
done will be necessary. The results given above have reference 
only to the old method of titration with a one-tenth normal solution 
of sodium hydrate. 

CHEMISTRY OF THE URINE. 

General Chemical Composition of the Urine. — A general idea 
of the chemical composition of the urine and the quantitative varia- 
tions of the individual components may be formed from the follow- 
ing table, which I have constructed from analyses made in my labor- 
atory. The individuals from which the urines were obtained were 
adults, and their general mode of life, as regards diet, exercise, etc., 
was that of the average American city-dweller. In addition, the 
following substances may be encountered under pathological con- 
ditions : serum-albumin, serum-globulin, albumoses, mucin (nucleo- 
albumin), glucose, lactose, inosit, dextrin, biliary constituents, viz., 
bile-acids and bile-pigments, blood-pigments, melanin, leucin, tyro- 
sin, oxybutyric acid, allantoin, fat, lecithin, cholesterin, acetone, 
alcohol, Baumstark's substance, urocaninic acid, cystin, hydrogen 
sulphide, and still others. 

1 The urine is carefully guarded against ammoniacal decomposition by the addition, 
to the first portion voided, of from 20 to 25 c.c. of a solution of 10 grammes of oil of 
peppermint to 100 c.c. of alcohol ; or, a few cubic centimeters of chloroform are added, 
which answer the same purpose. 
25 



386 



THE URINE. 



Analysis of Urine. 

Water . 1200 -1700 grammes. 

Solids . . . . 60.0 

Inorganic solids 25.0 -26.0 

Sulphuric acid (H 2 S0 4 ) • . . 2.0-2.5 

Phosphoric acid (P 2 5 ) 2.5-3.5 

Chlorine (NaCl) 10.0 -15.0 

Potassium (K 2 0) 3.3 

Calcium (CaO) . 0.2-0.4 

Magnesium (MgO) 0.5 

Ammonia (NH 3 ) 0.7 

Fluorides, nitrates, etc 0.2 

Organic solids 20.0 -35.0 

Urea 20.0 -30.0 

Uric acid 0.2-1.0 

Xanthin bases 1 .0 

Kreatinin ......... 0.05- 0.08 

Oxalic acid 0.05 

Conjugate sulphates 0.12- 0.25 

Hippuric acid 0.65- 0.7 

Volatile fatty acid 0.05 

Other organic solids 2.5 



Quantitative Estimation of the Mineral Ash of the Urine. — 

In order to estimate the amount of mineral ash in the urine the 
following method may be employed : 

Fifty c.c. of urine are evaporated to dryness in a weighed porce- 
lain dish, at a temperature of 100° C, and then heated, while 

Fig. 91. 




Desiccator. (W. Simon.) 



covered, over the free flame until gases cease to be evolved, care 
being taken not to heat too strongly in order to avoid sputtering. 
The residue is taken up with distilled boiling water, and, after 
standing, filtered through a Schleicher and Schiill's filter, the weight 
of the ash of which is known. The dish and the contents of the 
filter are well washed with hot water. Filtrate and washings are 
set aside and the dish and filter dried in the oven at 115° C. The 



CHEMISTRY OF THE URINE. 387 

filter is now placed in the dish and slowly incinerated. So soon as 
the ash has turned white the filtrate and washings are placed in the 
same dish, evaporated at 100° C, and then carefully heated over 
the free flame. Upon cooling in the desiccator (Fig. 91) the dish 
with its contents is weighed, the difference between its present and 
previous weight indicating the quantity of ash contained in 50 c.c. 
of urine. 

Precautions : 1. Care should be taken to allow the dish to be- 
come faintly red only for a moment, as some of the chlorine is 
otherwise volatilized. Some phosphoric acid may also escape, and 
too strong a heat, moreover, may cause the transformation of sul- 
phates into sulphides, the organic material present acting as a 
reducing agent. 

2. If the organic ash is not completely incinerated, it is best to 
allow the dish to cool and then to moisten the ash with a few drops 
of dilute sulphuric acid, when the heating is continued. 

The Chlorides. 

The chlorides which are excreted in the urine are derived from 
the food. As they are thus present in a much larger amount than 
all other inorganic salts combined, and in quantity more than suf- 
ficient to supply the needs of the body-economy, the relatively large 
amount of chlorides found in the urine under physiological condi- 
tions, as compared with the other inorganic constituents, is readily 
explained. 

Of the alkalies in the urine, sodium in combination with chlorine 
exists in greatest amount, and for clinical purposes it is most con- 
venient to calculate the total quantity of chlorides found in terms 
of sodium chloride ; a small proportion also occurs combined with 
potassium, ammonium, calcium, and magnesium. 

From 11 to 15 grammes of sodium chloride, representing the 
total quantity of chlorine, are normally eliminated in the twenty- 
four hours, the amount depending, of course, directly upon that 
contained in the food ingested. If the amount of nourishment is 
diminished, a decrease in the elimination of the chlorides is observed. 
If this is carried to the point of starvation, the chlorides disappear 
almost entirely from the urine, the traces remaining being derived 
from the body-fluids. The latter retain tenaciously a certain amount, 
which differs but slightly from that normally present. If at this 
stage food containing sodium chloride is again taken, a portion will 
be retained in the body until the original equilibrium is restored. A 
similar retention may be observed for a few days following the 
ingestion of large quantities of water, which causes an increased 
elimination of chlorides. 

This tenacity on the part of the body in retaining sodium chloride 



388 THE URINE. 

is strikingly seen when the potassium salt is substituted for the 
sodium salt ; in this case the amount of the sodium in the serum 
of the blood will be found to vary very slightly. 

It has also been shown that the excretion of sodium chloride 
can be increased very materially by the ingestion of potassium 
salts, notably the neutral potassium phosphate (K 2 HP0 4 ). This is 
supposed to decompose the sodium chloride present in the serum, 
resulting in the formation of potassium chloride and neutral sodium 
phosphate, which are both eliminated as foreign material ; a point 
is finally reached, however, when the sodium chloride ceases to be 
excreted. 

This provision of the economy, in virtue of which an increase in 
the elimination of the salt is followed by its retention, and a pre- 
vious retention by an increased elimination, is supposed to be refer- 
able to the albuminous metabolism taking place in the body. It 
may be stated, as a general rule, that any increase in the amount of 
circulating albumin will be followed by an increased elimination of 
chlorides, these having been previously retained by the albuminous 
bodies in consequence of the great affinity which exists between 
them. At the same time the elimination of the chlorides is influ- 
enced by the quantity of urine excreted, increasing and decreasing 
with its volume. 

Pathologically the excretion of the chlorides may vary within 
wide limits, diminishing on the one hand to zero and increasing 
on the other to 50 grammes or more in the twenty-four hours. A 
marked diminution, which in some cases may go on to a total 
absence, was formerly thought to be pathognomonic of acute croup- 
ous pneumonia. 1 More modern investigations, however, have shown 
that such a condition occurs to a greater or less degree in most acute 
febrile diseases, such as scarlatina, roseola, variola, typhus and 
typhoid fevers, recurrens, and acute yellow atrophy. 

The explanation of this phenomenon must be sought for, first, in 
a diminished ingestion of chlorides ; secondly, in a retention of these 
in the blood, Avhich probably is associated with an increase in the 
amount of the circulating albumin ; thirdly, in a diminished renal 
secretion of water ; fourthly, in a possible elimination of a portion 
of the chlorides through other channels, as in cases of severe diar- 
rhoea, the formation of serous exudates, etc. 2 Intermittent fever 
appears to be an exception to this rule ; usually it is true the 
chlorides are diminished, but not to the extent seen in the other 
diseases mentioned. They have, moreover, been found to increase 
during and sometimes immediately after a paroxysm, this increase 
being, of course, followed by a corresponding diminution. 

The chlorides are diminished in all acute and chronic renal dis- 

1 Kettenbacher, Wien. med. Zeit., 1850, p. 373. Heller, Heller's Archiv, 1844, vol. 
i. p. 23, 2 Salkowgki u. Leube, Lehre vom Harn, 1882, p. 174, 



CHEMISTRY OF THE URINE. 389 

eases associated with albuminuria, owing to some extent at least 
to a diminished secretion of water. 1 

In all cases of carcinoma of the stomach, and in chronic hyper- 
secretion associated with dilatation, a decrease is also observed, which 
in certain cases of hypersecretion and hyperacidity, the result of gas- 
tric ulcer, may go on to a total absence. 2 

In anaemic conditions the chlorides are likewise diminished, as 
also in rickets. In melancholia and idiocy a striking decrease is 
observed ; in dementia, chorea, and pseudohypertrophic paralysis 
this is less marked. A total absence has been noted in pemphigus 
foliaceus, and a considerable diminution in the beginning of impet- 
igo, as also in chronic lead poisoning. 

The chlorides are found in increased amount, on the other hand, 
in all conditions in which retention has previously occurred, chief 
among these being the acute febrile diseases and cases in which a re- 
sorption of exudates and transudates, associated with an increased 
diuresis, is taking place. A marked increase has also been noted in 
some cases of diabetes insipidus, in which 29 grammes have been 
eliminated in the twenty-four hours. 3 A similar increase may occur 
in prurigo, in which, in one instance, 29.6 grammes were passed in 
twenty-four hours. 4 In cases of general paresis, during the first 
stage, an increased elimination goes hand in hand with an in- 
creased ingestion of food. In epilepsy the polyuria following the 
attacks is associated with an increase in the chlorides. 

Of drugs, certain diuretics, and some of the potassium salts, as 
has been mentioned, produce an increase : the chlorine contained in 
chloroform, whether administered internally or as an anaesthetic, is 
in part excreted in the form of a chloride. Salicylic acid, on the 
other hand, is said to cause a temporary diminution. 

It is of practical importance to note that in acute febrile diseases 
the diminution in the chlorides appears to vary with the intensity 
of the disease, a decrease to 0.05 gramme pro die justifying the con- 
clusion that the case under observation is of extreme gravity. It 
may at times also indicate the previous occurrence of severe diarrhoea 
or the formation of exudates of considerable extent. A continued 
increase, on the other hand, should lead to the conclusion that the 
patient's condition is improving. 

The elimination of the chlorides also furnishes a fair index to the 
digestive powers of the patient. This rule also holds good for most 
chronic diseases. All other causes which might lead to an increase 
or decrease being eliminated, an excretion of from 10 to 15 grammes 
indicates a fair condition of the appetite and a normal digestive 
power, a decrease being associated with the reverse. 

1 Bohmann, Zeit. f. klin. Med., 1886, vol. i. p. 513. 

2 Gluzinski, Berlin, med. Woch., 1887, vol. xxiv. p. 983. 

3 Oppenheim. Zeit. f. klin. Med., vol. vi. 

4 v. Brueff, Wien. med. Woch., 1871, p. 552. 



390 THE URINE. 

An increased elimination of chlorides occurring in cases of oedema, 
and associated with the existence of serous exudates, is always of 
good prognostic omen, pointing to a resorption of the fluid. 

A continued elimination of more than 15 to 20 grammes, all other 
causes being excluded, may be considered as pathognomonic of dia- 
betes insipidus. 

Test for Chlorides in the Urine. — The recognition of the chlo- 
rides in the urine is based upon the fact that the addition of a solu- 
tion of silver nitrate causes their precipitation, the reaction taking 
place according to the equation 

AgN0 3 + NaCl = AgCl + NaN0 3 . 

The silver chloride thus formed is insoluble in nitric acid. 

The test is made in the following manner : after having removed 
any albumin that may be present, according to methods given else- 
where (see Albumin), a few cubic centimeters of urine are acidified, 
in a test-tube with about 10 drops of pure nitric acid, and treated 
with a few cubic centimeters of silver nitrate solution (1 : 20). 
The occurrence of a white precipitate indicates the presence of 
chlorides. An idea may be formed at the same time of the quantity 
present ; the occurrence of a heavy, caseous precipitate points to a 
large amount. 

Quantitative Estimation of the Chlorides by the Method of 
Salkowski-Volhard. 1 — When a solution of silver nitrate acidified 
with nitric acid is treated with a solution of potassium sulphocyanide 
or ammonium sulphocyanide, in the presence of a ferric salt, the 
potassium sulphocyanide first causes the precipitation of white silver 
sulphocyanide, which, like silver chloride, is insoluble in nitric acid : 

AgN0 3 + KSCN = AgSCN -f KN0 3 . 

As soon as every trace of silver is precipitated, it combines with 
the ferric salt to form ferric sulphocyanide, which is of a blood-red 
color : 

6KSCN + Fe 2 (S0 4 ) 3 = Fe 2 (SCN) 6 + 3K 2 S0 4 . 

If the potassium sulphocyanide solution is of known strength, it 
is possible to estimate accurately the amount of silver present in the 
solution, the ferric salt serving as an indicator of the end of the re- 
action between the silver and the potassium sulphocyanide. 

Application to the urine : to urine which has been acidified with 
nitric acid an excess of a silver solution of known strength is added, 
and the silver not used in the precipitation of the chlorides then esti- 
mated as indicated above. The difference between the quantity thus 
found and the total amount used will be that consumed in the pre- 
1 E. Salkowski, Zeit. f. physiol. Chem., vol. i. p. 16, and vol. ii. p. 397. 



CHEMISTRY OF THE URINE. 391 

cipitation of the chlorides, from which, knowing the strength of the 
silver solution, its equivalent in terms of sodium chloride is readily 
determined. 

Reagents required : 

1. A solution of silver nitrate of such strength that each cubic 
centimeter shall correspond to 0.01 gramme of sodium chloride. 

2. A solution of potassium sulphocyanide of such strength that 
25 c.c. shall correspond to 10 c.c of the silver nitrate solution. 

3. A solution of a ferric salt, such as ammonio-ferric alum, satu- 
rated at ordinary temperature. 

4. Nitric acid (specific gravity 1.2). 
Preparation of these solutions : 

1. As pointed out, the silver nitrate solution is made of such 
strength that each cubic centimeter shall correspond to 0.01 gramme 
of sodium chloride ; in other words, a standard solution is employed. 

The silver nitrate must be pure, and it is best to use the crystal- 
lized salt, and not the sticks wrapped in paper, which always contain 
reduced silver. In order to test the purity of the salt, about 1 
gramme is dissolved in distilled water, heated to the boiling-point, 
the silver precipitated by dilute hydrochloric acid and filtered off. 
When evaporated in a platinum crucible the filtrate should leave 
either no residue at all or only a very faint one ; otherwise it is 
necessary to recrystallize the salt until the desired degree of purity 
is reached. 

The determination of the quantity to be dissolved in 1000 c.c. of 
water is based upon the fact that 1 molecule of silver nitrate 
(molecular weight 170) combines with 1 molecule of sodium 
chloride (molecular weight 58.5) to form silver chloride and sodium 
nitrate. As the solution of silver nitrate shall be of such strength 
that 1 c.c. corresponds to 0.01 gramme of sodium chloride, or 1000 
c.c. to 10 grammes, the quantity to be dissolved in 1000 c.c. is found 
according to the following equation : 

58.5 : 170 : : 10 : x, 58.5 x = 1700, x = 29.059. 

Theoretically, then, this quantity should be dissolved in 1000 c.c. 
of water. It is better, however, to dissolve it in a quantity some- 
what less than 1000 c.c, such as 900 or 950 c.c, as the silver salt 
contains water of crystallization and the weigh ed-ofF quantity would 
not represent the exact amount required, but less, the correcting of 
a solution which is too strong being a much simpler matter than 
that of a solution which is too weak. 

To make this correction, or, in other words, to bring the solution 
to its proper strength, 0.15 gramme of sodium chloride which has 
previously been dried carefully by heating in a platinum crucible, is 
accurately weighed off, dissolved in a little distilled water, and further 
diluted to about 100 c.c To this solution a few drops of a solution 



392 THE URINE. 

of potassium chromate are added, when the mixture is titrated witn 
the silver solution. 

The silver nitrate will first precipitate the sodium chloride, and 
then combine with the potassium chromate, forming red silver 
chromate, according to the equation 

2AgN0 3 + K 2 Cr0 4 = Ag 2 Cr0 4 + 2KN0 3 . 

The slightest orange tinge remaining after stirring indicates the 
end of the reaction. Were the solution of the silver nitrate of the 
proper strength, exactly 1 5 c.c. should have been used, as each cubic 
centimeter shall represent 0.01 gramme of sodium chloride. As a 
matter of fact, less will in all probability be needed, the solution 
having been purposely made too strong. Its correction then be- 
comes a simple matter, as it is merely necessary to determine the 
degree of dilution required. 

Supposing that 29.059 grammes of silver nitrate were dissolved 
in 900 c.c. of water, and that 14.5 c.c. instead of 15 c.c. had 
been required to precipitate the 0.15 gramme of sodium chloride, 
it is evident that each 14.5 c.c. of the remaining solution must 
be diluted with 0.5 c.c. of water. It is, hence, only necessary to 
divide the number of cubic centimeters of the silver nitrate solution 
remaining by 14.5; the result multiplied by 0.5 represents the 
amount of water which must be added in order to bring the solution 
to the required strength. Hence the rule for the correction of a 
solution which has been found too strong : 

n 

in which C represents the number of cubic centimeters of water 
which must be added to the solution remaining ; N the total number 
of cubic centimeters remaining after titration ; n the number of 
cubic centimeters consumed in one titration ; and d the difference 
between the number of cubic centimeters theoretically required and 
that actually used in one titration. 

In the example given the equation would then read : 

936.5 X 0-5 ^ 32 29j 
14.5 

32.29 c.c. of distilled water are added to the remaining 936.5 c.c, 
when the strength of the solution is tested by a second titration. If 
the solution is found too weak, it is best to make it too strong, and 
then to correct as described. 

2. Preparation of the potassium sulphocyanide solution : from 
the equation AgNO a + KSCN = AgSCN + KN0 3 , it is seen that 
1 molecule of silver nitrate (molecular weight 170) combines with 
1 molecule of potassium sulphocyanide (molecular weight 97). The 



CHEMISTRY OF THE URINE. 393 

quantity of the latter to be dissolved in 1000 c.c. of water is then 
found from the following equation : 

170 : 97 : : 11.6236 : x ; 170 x = 11.6236 X 97 ; x = 6.6. 

As potassium sulphocyanide is extremely hygroscopic, a solution 
is made which is too strong, by dissolving about 10 grammes of the 
salt in 900 c.c. of distilled water. In order to bring this solution to 
its proper strength, 10 c.c. of the silver solution are diluted to 100 
c.c; I c.c. of nitric acid (specific gravity 1.2) and 5 c.c. of the am- 
monio-ferric alum solution are added, when the mixture is titrated 
with the potassium sulphocyanide solution ; the end-reaction is 
recognized by the production of a slightly reddish color, which per- 
sists on stirring. The sulphocyanide solution having been purposely 
made too strong, it will be found that less than 25 c.c. are needed to 
precipitate all the silver present. The quantity of water necessary 
for dilution is ascertained, as above, according to the formula 

n 

3. The solution of ammonio-ferric alum is a solution saturated at 
ordinary temperatures, care being taken to insure the absence of 
chlorides in the salt, which may be effected, if necessary, by recrys- 
tallization. 

Method as Applied to the Urine. — Ten c.c. of urine are placed in a 
small stoppered flask bearing a 100 c.c. mark, diluted with 50 c.c. 
of distilled water, and acidified with 4 c.c. of nitric acid. From a 
burette 15 c.c. of the standard solution of silver nitrate are added. 
The mixture is thoroughly agitated and diluted with distilled water 
to the 100 c.c. mark. The silver chloride formed is filtered off 
through a dry folded filter into a dry graduate ; 80 c.c. of the 
filtrate are placed in a beaker, and, after the addition of 5 c.c. of the 
ammonio-ferric alum solution, titrated with the sulphocyanide solu- 
tion until the end-reaction — i. e., a slightly reddish tinge — is seen. 
If necessary, two such titrations should be made, the sulphocyanide 
solution being added 1 c.c. at a time in the first, while in the second 
the total number of cubic centimeters needed to bring about the 
end-reaction, less 1 c.c, are added at once, and then 0.1 c.c at a 
time. 

The amount of chlorides present in the urine is calculated as fol- 
lows : 

Example. — Total quantity of urine 600 c.c. ; 6.5 c.c of the 
sulphocyanide solution were required to bring about the end-reaction 
in 80 c.c. of the filtrate; this would correspond to 8.125 c.c. for 
the total 100 c.c of filtrate, representing 10 c.c of urine, as is seen 
from the equation 

n:80::z:100; 80z = 100n; x-=™0ji=5ji, 

80 4 



394 THE URINE. 

in which x represents the number of cubic centimeters corre- 
sponding to 100 c.c. of the nitrate, and n the number of cubic 
centimeters actually used. 

These 8,125 c.c. were used in precipitating the silver nitrate not 
decomposed by the chlorides. As 25 c.c. of the sulphocyanide 
solution correspond to 10 c.c. of the silver solution, the excess of 
silver solution in cubic centimeters is found from the equation 

25:10::*:*; 25"* = 10*; x = 1 M-=^, 

25 5 

in which x represents the excess of the silver solution in cubic 
centimeters, and N that of the sulphocyanide solution as found 
according to the equation above, x in this case being 3.25 c.c. 

The difference between the total amount of silver solution em- 
ployed (i.e., 15 c.c.) and the excess (i.e., 3.25 c.c.) indicates the 
number of cubic centimeters necessary for the precipitation of 
the chlorides in 10 c.c. of urine. In the case under consideration 
11.75 c.c. were employed. As 1 c.c. of the silver solution repre- 
sents 0.01 gramme of sodium chloride, there must have been present 
in the 10 c.c. of urine 0.1175 gramme; in 100 c.c, hence, 1.175 
grammes, and in the total amount — i. e., 600 c.c. of urine — 7.05 
grammes. 

From these considerations the following short rule results : instead 
of first multiplying the number of cubic centimeters of the potas- 
sium sulphocyanide solution corresponding to 80 c.c. of the filtrate, 
by |-, and the result by |-, in order to find the number of cubic 
centimeters of the potassium sulphocyanide solution representing 
the excess of silver nitrate in 100 c.c. of the filtrate, and then 
deducting the result from 15, it is simpler to multiply by \ directly 
and deduct the result from 15, the number of grammes of sodium 
chloride contained in 1000 c.c. of uriue being thus found. This 
figure is then corrected for the total amount of urine. 

The method described may be employed in the presence of albu- 
mins, album oses, and sugar ; the urine, however, must be fresh, so 
as to insure the absence of nitrous acid. 

Direct Method. 1 — If absolute accuracy is not required, the fol- 
lowing method may be employed : 

Ten c.c. of urine are diluted with distilled water to 100 c.c. and 
treated with a few drops of a solution of potassium chromate. This 
mixture is titrated with a one-tenth normal solution of silver nitrate 
until the end-reaction is reached — i. e., a faint orange tinge — which 
no longer disappears on stirring. The number of cubic centi- 
meters used multiplied by 0.01 will indicate the amount of chlorides 
present in 10 c.c. of urine. 

As uric acid, the xan thin-bases, hyposulphites, sulphocyanides, 

1 F. Mohr, Lehrbucli d. Titrirmethode, 1856, ii. p. 13. 



CHEMISTRY OF THE URINE. 395 

and pigments are also precipitated by the silver nitrate, the end- 
reaction is delayed ; moreover, unless the urine is very pale, its 
recognition may be difficult, and the error thus caused considerable. 
This is especially true of febrile urines which contain only a small 
amount of chlorides. 

Should iodides or bromides have been taken, these must first be 
removed, as silver iodide and bromide, which are insoluble in nitric 
acid, would give too high a value. 

To this end, the following method, which is also a very accurate 
one, should be employed, its only disadvantage being the amount of 
time required. 

Estimation of the Chlorides after Incineration (according to 
Neubauer and Salkowski). 1 — The principle of this method is the 
destruction of all organic material and the subsequent estimation of 
the chlorides contained in the mineral ash by one of the methods 
described. Ten c.c. of urine are evaporated to dryness in a platinum 
crucible at a temperature slightly below 100° C, after the addition 
of a little pure dried sodium carbonate and from 3 to 5 grammes 
of potassium nitrate. The addition of the sodium carbonate insures 
the conversion of any ammonium chloride which may be present 
into sodium chloride ; the potassium nitrate acts merely as an oxi- 
dizing agent. The residue is now carefully heated at a moderate tem- 
perature, allowed to cool, dissolved in distilled water, and accurately 
neutralized with very dilute nitric acid. In this solution the chlorides 
are estimated most conveniently according to the second method. 

Should iodides or bromides be present, the aqueous solution just 
referred to is acidified with sulphuric acid, and the iodine and 
bromine thereby liberated extracted w T ith carbon disulphide. As 
complete removal of these bodies is, however, only possible in the 
presence of a nitrite, it is better not to rely upon the presence of 
any that may have been formed during the process of incineration, 
but to add a few drops of a solution of potassium nitrite. After 
extraction the nitrous acid is decomposed by the addition of a little 
urea. The solution is then neutralized with sodium carbonate ; 
should it be alkaline, dilute acetic acid is added until neutral. In 
this solution the chlorides are most conveniently estimated according 
to the second method. 

Albumin and sugar, if present, should be removed before the 
addition of the sodium carbonate and potassium nitrate, so as to 
obviate losses from sputtering, which otherwise would occur. Nitrous 
acid must also be removed for reasons given above. 

The Phosphates. 

The phosphates occurring in the urine are sodium, potassium, cal- 
cium, and magnesium salts of the tribasic acid H 3 P0 4 . The most 

1 E. Salkowski, Pfluger's Archiv, vol. vi. p. 214. 



396 THE URINE. 

important of these, as was pointed out in the chapter on Reaction, 
is the diacid sodium phosphate NaH 2 P0 4 , to which the acidity of 
the urine is due. It is owing to the presence of this salt in the 
urine that the calcium phosphate is held in solution ; the fact, at 
least, that calcium and magnesium phosphates are thrown down 
when the urine is neutralized, would point to this conclusion. 

The composition of the phosphates is liable to considerable varia- 
tion, depending upon the degree of acidity of the urine. As would 
be expected, diacid sodium phosphate and diacid calcium phosphate 
are present in an acid urine ; in an amphoteric urine, in addition to 
these there are found disodium phosphate, monocalcium phosphate, 
and monomagnesium phosphate, while in an alkaline urine trisodic 
phosphate, neutral calcium phosphate, and neutral magnesium phos- 
phate may be present. 

The alkaline phosphates normally exceed the earthy phosphates 
by one-third, and sodium is combined with far the greater amount 
of phosphoric acid, the potassium salt normally occurring in only 
very small amounts. 

In addition to the mineral phosphates, phosphoric acid is excreted 
also in combination with glycerin as glycerin-phosphoric acid, which 
need not, however, be considered in a quantitative estimation, as it 
is present only in traces. 1 

As in the case of the chlorides, the greater portion of the phos- 
phates is derived from the food, while only a small portion is refer- 
able to the phosphorus built up in the proteid molecule, be this in 
the form of a muscle-cell, a nerve-cell, a red blood-corpuscle, or bone. 
But just as the percentage of sulphur varies in the different tissues, 
so also does that of phosphorus vary ; nerve-tissue, for example, 
which is very rich in lecithin and nucleins, yields relatively more 
phosphorus than muscle-tissue. 

Not all the phosphoric acid ingested, however, is excreted in the 
urine, as one-third to one-fourth of the total quantity is eliminated 
in the feces. 

The quantity of phosphoric acid excreted, which normally varies 
between 2.5 and 3 grammes, is thus largely dependent upon the 
amount ingested, increasing with an animal and decreasing with a 
vegetable diet. 2 During starvation a considerable increase is like- 
wise observed, referable, no doubt, to an increased destruction of bony 
tissue, which is very rich in the phosphates of the alkaline earths. 
In accordance with this view, increased amounts of calcium and 
magnesium are also seen during starvation. The relation between 
the excretion of phosphoric acid and nitrogen, normally 1 : 7, changes, 
moreover, in such a manner that both the absolute and the relative 
amount of phosphoric acid, as compared with the nitrogen, increases ; 

1 Lepine et Eymonnet, Compt. rend, de la Soc. de Biol., 1882. 

2 Ziilzer, Virchow's Archiv, vol. lxvi. p. 223. 



CHEMISTRY OF THE URINE. 397 

this leads to the conclusion that in addition to the muscles some 
other tissue rich in phosphorus and relatively poor in N must suffer 
during the process, and the only one which could enter into con- 
sideration is bone. 1 If at this time food containing phosphorus is 
again given, a retention will take place, so that the general rule 
stated in the chapter on Chlorides, that increased elimination is fol- 
lowed by a certain degree of retention, and that a previous retention 
is followed by an increased elimination, seems to hold good for all the 
mineral acids found in the urine (see also the chapter on Sulphates). 

An increased elimination is caused also by the ingestion of large 
quantities of water, which is followed by a certain degree of retention. 

Observations on the phosphatic excretion during muscular exercise 
have not given uniform results. 2 Mental exercise appears to cause a 
diminished excretion of the alkaline phosphates and an increased 
elimination of the earthy phosphates. 3 The latter also takes place 
during sleep. 

In disease the total amount of phosphates may either be increased 
or diminished. 

A diminished elimination is observed in most cases of acute febrile 
disease, such as pneumonia, typhoid fever, typhus fever, recurrens, 
during a paroxysm of intermittent fever, etc. The degree of dimi- 
nution is usually proportionate to the severity of the disease, reaching 
its lowest figure as death approaches. Such a state of affairs may, 
at first sight, appear paradoxical in view of what has been said above 
of the effects of tissue-destruction upon the elimination of phos- 
phates. It is necessary, however, to distinguish sharply between an 
increased production and an increased elimination ; in all probability 
a retention occurs analogous to that of the chlorides, which may be 
observed under the same conditions. It has been supposed that the 
phosphates set free during the process of tissue-destruction are 
utilized in the building up of new leucocytes, and an increase in 
these is actually noted in some of the diseases mentioned. A dimin- 
ished excretion of phosphates is, however, not always observed, 
and an increased elimination may occur in certain cases. In fatal 
cases this condition may persist even until the time of death. It 
is very difficult to give a satisfactory explanation of this fact at 
the present time. The phenomenon, in typhoid fever at least, 
appears to be connected with the intensity of the nervous manifesta- 
tions, and Eobin concludes that here an increased elimination during 
the fastigium is an unfavorable omen, while an increase during defer- 
vescence warrants a favorable prognosis. A similar decrease in the 
phosphates has also been observed in pulmonary phthisis associated 
with high fever. 4 

1 Zulzer, loc. cit. 

2 Fleischer u. Penzoldt, Virchow's Archiv, vol. lxxxvii. p. 210, 

3 Mairet, Corupt. rend, de la Soc. de Biol., 1884. 

* Bdlefsen, Schmidt's Jahresber., vol. cxcvi. p. 59, 



398 THE URINE. 

Very interesting and important is the diminished excretion of 
the phosphates associated with acute and, to some extent also, with 
chronic nephritis, amyloid degeneration of the kidneys, and the 
anaemias, in which an actual insufficiency on the part of the kidneys 
in the elimination of these salts appears to exist. 1 

A diminished or, at least, no increased excretion is seen in certain 
diseases of the bones, such as osteomalacia, although an increase in 
the earthy phosphates has been noted. This may depend either upon 
a retention or an elimination through other channels. The earthy 
phosphates especially are found in greatly diminished amount, or 
may even be absent altogether in certain cases of nephritis. A 
similar condition is observed in acute and chronic rheumatism. 

The data regarding the phosphatic elimination iu nervous and 
mental diseases are, on the whole, scanty and by no means uniform. 

During attacks of hysteria major, in contradistinction to epilepsy, 
in which an increased elimination takes place, the phosphates are 
diminished, the degree of diminution being generally proportionate 
to the intensity of the attack, increasing again together with the other 
urinary constituents with the subsequent increase in the diuresis. 2 

In chronic lead poisoning a diminution to one-third of the normal 
quantity may occur. Very low figures have been noted in Addison's 
disease, in acute yellow atrophy (in which even a total absence may 
occur), and in certain cases of hepatic cirrhosis. In gout the phos- 
phoric acid curve follows that of the uric acid quite closely, decreas- 
ing before the onset of the acute symptoms and then rising and 
reaching its maximum about the third day (see Uric Acid). 3 

An increased elimina tion of phosphates, on the other hand, amount- 
ing in some cases to 1 or even to 9 grammes in the twenty-four 
hours, has been described by Teissier, of Lyon, under the name of 
phosphatic diabetes, the patient presenting various symptoms com- 
monly seen in diabetes mellitus ; sugar, however, is usually absent. 
Whether or not phosphatic diabetes is a disease sui generis is not 
certain. 4 

In true diabetes mellitus a curious relation has been found to 
exist between the elimination of sugar and of phosphates, the quan- 
tity of the latter rising and falling in an inverse ratio to the amount 
of sugar. In diabetes insipidus a slight increase is at times found. 

Corresponding to the phosphatic retention observed in acute febrile 
diseases an increased elimination is noted during convalescence. An 
increase occurs in the course of cerebrospinal meningitis. 

In a case of pseudoleukemia an increase of 7 grammes has been 
noted, while the number of red corpuscles fell from 2,200,000 to 
800,000 in four days. To judge from the very careful observations 

1 Fleischer, Deutsch. Arch. f. klin. Med., vol. xxix. p. 129. 

2 De la Tourette and Cathelineau, Centralbl. f. d. med. Wiss., 1889, vol. xlviii. p. 872. 

3 T. B. Futcher, Jour. Am. Med. Assoc, 1902, vol. xxxix. p. 1046. 

i G. Rankin, " Phosphatic Diabetes," Lancet, March 24, 1900. Teissier, These, 
Paris, 1876, 



CHEMISTRY OF THE URINE. 399 

made, there could be no doubt that the high degree of phosphaturia, 
which was limited to the alkaline phosphates, was referable to this 
latter source. In leukaemia also an increase to 7 grammes has been 
observed on the day preceding death ; commonly, however, the in- 
crease is but slight in this disease. 1 

While it is apparent that important conclusions cannot be drawn, 
on the whole, from a knowledge of the absolute phosphatic elimina- 
tion, unless it be from a study of the relation existing between the 
excretion of the alkaline and earthy phosphates, a study of the rela- 
tive phosphatic excretion seems to promise more valuable results. 
According to Zulzer, 2 a definite amount of the phosphates and of the 
urinary nitrogen is referable to the destruction of albuminous mate- 
rial, so that the relation between the phosphoric acid and the nitro- 
gen must be constant. Another portion, however, is derived from 
lecithin, one of the most important constituents of nerve- tissue, 
which contains more phosphorus than the albuminous molecule. 
Whenever, then, the lecithin-containing tissues are more involved 
in the general metabolism than under normal conditions the rela- 
tion will no longer be a stable one. This relation which exists 
between the elimination of nitrogen and phosphoric acid has been 
termed the relative value of phosphoric acid. 

The relative value of phosphoric acid in the urine has been calcu- 
lated as varying from 17 to 20, that of the blood being 3, of muscle- 
tissue 12.1, of brain 44, of bone 426 to 430. This value supposes 
the absolute value to vary between 2 and 3 grammes pro die. It is 
found according to the following equation : 

100 . P 9 (\ 



N : P,(X : : 100 : x\ and x = 



N 



in which N indicates the amount of nitrogen actually observed, 
P 2 O s the amount of phosphoric acid in the same specimen of urine, 
and x the amount of P 2 O s corresponding to 100 grammes of K. 
By observing this relative value a much better idea may be formed 
of the metabolic processes taking place in the body in disease than 
from a mere expression of the absolute phosphatic value. 

In acute febrile diseases the relative as well as the absolute dimi- 
nution of the phosphates has been ascribed to a retention, they being 
possibly utilized in the building up of white blood-corpuscles. In 
the course of these diseases oscillations in the relative value are fre- 
quently observed ; during convalescence the relative as well as the 
absolute value again rises. 

In accordance with these considerations a diminished relative ex- 
cretion of phosphoric acid should be expected in all cases associated 
with a notable elimination of leucocytes through other channels, as 
in pneumonia, for example, or a storing away of the same, as in 

1 Fleischer u. Penzoldt, loc. cit. 2 Loc. cit. 



400 THE URINE. 

cases of empyema. The facts observed are in accord with this 
view. 

A relative decrease has further been noted in the various forms of 
anaemia, conditions of cerebral excitation, and especially preceding 
an attack of epilepsy. In progressive paralysis following syphilis 
the relative value, at first low, rises greatly after the administration 
of potassium iodide, while the excretion of the earthy phosphates is 
lessened. In chronic cerebral affections, delirium tremens, and acute 
hydrocephalus a relative decrease has been noted. In mania, during 
the period of excitement, both the alkaline and the earthy phosphates 
are found increased, while during the stage of depression, as also in 
melancholia, the alkaline phosphates are diminished and the earthy 
phosphates increased. On the other hand, an increase in the relative 
value has been noted in apoplexy (amounting to 34.3 in one case, 
two days after an attack), brain tumors, tabes, arthritis deformans 
(30), pernicious anaemia (23.8—58), etc. 1 

Of drugs, bromides appear to diminish the absolute amount of 
phosphoric acid. Cocain and quinin cause a decrease, and salicylic 
acid an increase. A relative decrease is produced by the cerebral 
excitants, such as strychnin, small doses of alcohol, phosphorus, 
valerian, cold baths, salt-water baths, etc. An opposite effect is 
produced by the cerebral depressants, such as chloroform, morphin, 
chloral, large doses of alcohol, potassium bromide, mineral and 
vegetable acids, prolonged cold baths, Turkish baths, low tempera- 
ture, etc. 

Tests for the Phosphates in the Urine. — The test for the detec- 
tion of the phosphates occurring in the urine depends upon the pre- 
cipitation of phosphoric acid by means of ferric chloride as ferric 
phosphate, which is insoluble in cold acetic acid : 



or 



2NaH 2 P0 4 + Fe 2 Cl 6 = Fe 2 (P0 4 ) 2 + 2NaCl + 4HC1, 
2Na 2 HPO, + Fe 2 Cl 6 = Fe 2 (P0 4 ) 2 + 4NaCl + 2HC1. 



The same result may be accomplished by the addition of a solution 
of uranyl nitrate ; this gives rise to the formation of uranyl phos- 
phate, which is also insoluble in acetic acid : 

Na 2 HP0 4 + 2UO.NO3 = 2NaN0 3 + (UO) 2 HP0 4 , 
Na 2 HP0 4 + UO.NO3 = NaNO + UO.H 2 P0 4 . 

Test. — A few cubic centimeters of urine are acidified with a few 
drops of acetic acid, and treated with a few drops of a solution of 
ferric chloride (1 part of the officinal solution to 10 parts of water), 
when the occurrence of a yellowish- white precipitate will indicate 
the presence of phosphates. 

1 Zulzer u. Edlefsen, loc. cit. 



CHEMISTRY OF THE URINE. 401 

If a solution containing an acid phosphate of the alkalies is treated 
with an alkaline hydrate, the diacid alkaline phosphate is trans- 
formed into the monacid salt, according to the equation 

NaH 2 P0 4 + NH 4 OH = NaNH 4 HP0 4 + H 2 0. 

This is further changed into the normal salt, as represented : 

3NaNH 4 HP0 4 + NH 4 OH = Na 3 P0 4 + (NH 4 ) 3 (P0 4 ) 2 + H 2 0. 

As the monacid and neutral salts are both readily soluble, the 
solution remains clear. If at the same time, as in the urine, a 
soluble diacid phosphate of the alkaline earths is present, this like- 
wise is transformed into the monacid and finally into the neutral 
salt ; the latter, however, being insoluble, is thrown down : 

(1) Ca(H 2 P0 4 ) 2 + 4NH 4 OH = Ca(NH 4 ) 2 (P0 4 ) 2 + 4H 2 0. 

(2) 3Ca(NH 4 ) 2 (P0 4 ) 2 = Ca 3 (P0 4 ) 2 + 2(NH 4 ) 3 P0 4 . 

Test for the Earthy Phosphates. — Ten c.c. of urine are 
rendered alkaline with ammonia, when the occurrence of a flocculent 
precipitate will indicate their presence. 

Test for the Alkaline Phosphates. — After having removed 
the earthy phosphates from 10 c.c. of urine, as just described, the 
clear filtrate is acidified with acetic acid and tested with ferric chlo- 
ride or uranyl nitrate, as shown above. 

The alkaline phosphates may also be detected by treating the 
ammoniacal filtrate with a few drops of magnesia mixture (1 part of 
crystallized magnesium sulphate, 2 parts of ammonium chloride, 4 
parts of ammonium hydrate, and 8 parts of distilled water), when 
ammonio-magnesium phosphate, which is almost insoluble in ammo- 
nium hydrate, will be thrown down. The reaction takes place be- 
tween the monacid or neutral sodium phosphate and the magnesium 
sulphate according to the equation 

Na 2 HP0 4 + MgS0 4 + NH 4 OH + NH 4 C1 = MgNH 4 P0 4 + NH 4 C1 + Na 2 S0 4 + H 2 0. 

Quantitative Estimation of the Total Amount of Phosphates. 

— Principle. — When a solution of disodium phosphate acidified with 
acetic acid is treated with a solution of uranyl nitrate or uranyl 
acetate, a dirty-looking, white precipitate of uranyl phosphate is 
thrown down, which is formed according to the equation given 
above. It is apparent that the quantity of phosphoric acid can be 
estimated accurately, if the solution of uranyl nitrate or acetate is of 
known strength. 

Solutions required : 

1. A solution of uranium nitrate of such strength that 20 c.c. 
shall correspond to 0.1 gramme of P 2 5 . 

26 



402 THE URINE. 

2. A solution containing sodium acetate and acetic acid. 

3. Tincture of cochineal. 
Preparation of these solutions : 
1. From the equation 

2UO.N0 3 -f Na 2 HP0 4 = (UO) 2 HP0 4 + 2NaN0 3 

it is apparent that 2 molecules of uranium nitrate combine with 1 
molecule of disodium phosphate to form uranium phosphate and 
sodium nitrate. The molecular weight of uranium nitrate being 
318 and that of disodium phosphate 142, it is seen that 636 parts 
by weight of the former combine with 142 parts by weight of the 
latter. 

As 20 c.c. of the solution of uranium nitrate shall correspond to 
0.1 gramme of P 2 5 , 1000 c.c. must be equivalent to 5 grammes of 
P 2 5 . In 142 parts by weight of disodium phosphate there would 
be present 71 grammes of P 2 5 , equivalent to 636 parts by weight 
of uranium nitrate. The quantity of the latter, then, to be dis- 
solved in 1000 c.c. of water will be found from the equation : 636 : 
71 : : x : 5 ; and x = 44.78. 

44.78 grammes of uranium nitrate are weighed off and dissolved 
in about 900 c.c. of water, the solution being purposely made too 
strong for reasons pointed out in the chapter .on Chlorides. In 
order to bring this solution to its proper strength it is necessary to 
titrate with the uranium solution a solution of disodium phosphate 
of such strength that each 50 c.c. shall contain 0.1 gramme of P 2 5 , 
or 1000 c.c. 2 grammes. The molecular weight of Na 2 HP0 4 -f 
12H 2 being 358, this amount of disodium phosphate in grammes 
is equivalent to 1 42 grammes of P 2 0- ; the quantity of P 2 5 cor- 
responding to 2 grammes, in terms of Na 2 HP0 4 + 1 2H 2 0, is found 
from the equation : 358 : 142 : : x : 2 ; and x = 5~042. This 
amount of pure, dry, and non-deliquescent Na 2 HP0 4 is dissolved in 
1000 c.c. of distilled water. If non-deliquescent disodium phos- 
phate is not at hand, about 6 or 7 grammes of the salt are dissolved 
in 1000 c.c. of distilled water ; of this solution 50 c.c. are evapo- 
rated in a weighed platinum dish, and the residue gently heated, the 
disodium phosphate being thereby transformed into sodium pyro- 
phosphate, Na 4 P 2 O r , according to the equation 

2Na 2 HP0 4 = Na 4 P 2 7 + H 2 0. 

The molecular weight of Na 4 P 2 7 being 266, this corresponds to 
142 grammes of P 2 O s . If the solution is of the correct . strength 
— i. e,, containing 0.1 gramme of P 2 O s in 50 c.c. of water — the 
residue should weigh 0.1873 gramme, as is seen from' the equation : 
132 : 266 : : 0.1 : x ; and x = 0.1873. Supposing, however, that the 
residue weighs 0.1921 gramme, it is manifest that the solution is 



CHEMISTRY OF THE URINE. 403 

too strong, and must be diluted, the degree of dilution being ascer- 
tained according to the equation : 0.1873 : 1000 : : 0.1921 : x; and 
x = 1025 ; i. e., 1000 c.c. of the solution must be diluted to 1025 
c.c. to make it of the proper strength. 

In the case given, 50 c.c. were used ; the 950 c.c. are then diluted 
with the amount of water found from the equation : 1000 : 1025 : : 
950 : x ; and x = 973.75. Having thus obtained a solution of diso- 
dium phosphate of such strength that each 50 c.c. shall contain 0.1 
gramme of P 2 5 , this is titrated with the uranium solution, which 
has been made too strong, in order to determine the amount of 
water that must be added to the latter. To this end, a burette is 
filled with the uranium solution ; 50 c.c. of the disodium phosphate 
solution are treated with a few drops of the tincture of cochineal 
and 5 c.c. of the acetic acid mixture (see below). This mixture is 
heated in a beaker, and as soon as the boiling-point has been reached 
titrated with the uranium solution until a trace of a greenish color 
is noticed in the precipitate which does not disappear on stirring. 
This point having been accurately determined by means of a second 
titration, the number of cubic centimeters of distilled water with 
which the remaining solution must be diluted is determined accord- 

N. d 
ing to the formula : (7= — '- — , in which C represents the number 

n 

of cubic centimeters which must be added, N the number of cubic 
centimeters remaining after the test-titration, n the number of cubic 
centimeters consumed in one titration to bring about the end- 
reaction, and d the difference between the number of cubic centi- 
meters used in one titration and that theoretically required. The 
amount of distilled water necessary for dilution is now added and 
the solution again tested, when 20 c.c. will correspond to 0.1 gramme 
of P 2 0, 

2. The acetic acid mixture is prepared by dissolving 100 grammes 
of sodium acetate in a little water, adding 30 grammes of glacial 
acetic acid and diluting the whole to 1000 c.c. 

3. Tincture of cochineal. This may be prepared as follows : a 
few grammes of cochineal granules are digested at ordinary tempera- 
tures with 250 c.c. of a mixture of 3 volumes of water and 1 volume 
of 94 per cent, alcohol. The solution is then decanted and ready 
for use. The residue may be utilized in the preparation of a fresh 
supply of the tincture. 

Application to the Urine. — Fifty c.c. of clear filtered urine are 
treated with 5 c.c. of the acetic acid mixture, the object being to 
transform any monacid sodium phosphate present into diacid sodium 
phosphate, and to neutralize any nitric acid that may be formed 
during the titration, as otherwise the nitric acid would cause a partial 
solution of the precipitated uranyl phosphate. A few drops of the 
tincture of cochineal are added, when the mixture is heated to the 



404 THE URINE. 

boiling-point and titrated as described above. Two titrations are 
usually required. 

Should it be desired to use potassium ferrocyanide as an indicator, 
the uranium solution must have been standardized with the same 
indicator, as errors will otherwise arise. The technique is simple. 
A number of drops of the potassium ferrocyanide solution (about 
5 per cent.) are placed on a porcelain plate. After every addition 
of the uranium solution to the boiling urine a drop of the mixture 
is mixed on the plate with the ferrocyanide drop. The end reaction 
is indicated by the occurrence of a brown color. 

The results are calculated as follows : supposing 15 c.c. of the 
uranium solution to have been used, the corresponding amount of 
P 2 O s in 50 c.c. of urine is found from the equation : 20 : 0.1 : : 15 : x ; 
and x = 0.075. The percentage-amount would, hence, be 0.075 X 
2 == 0.15. Supposing the total amount of urine to have been 2000 
c.c, the elimination of P 2 O s would correspond to 3 grammes. 

The presence of sugar and albumin does not interfere with the 
method. 

Separate Estimation of the Earthy and Alkaline Phosphates. 
— If the alkaline and earthy phosphates are to be determined sepa- 
rately, the total amount of P 2 0. is estimated in one portion of the 
urine, while the P 2 5 in combination with the alkaline earths is 
determined in another, as follows : 

Two hundred c.c. of filtered urine are made strongly alkaline with 
ammonium hydrate and set aside, covered, for several hours, when 
the earthy phosphates thus precipitated are collected on a filter, 
washed with dilute ammonia (1 : 3), and then transferred to a beaker, 
with the aid of a little water containing a few drops of acetic acid, 
by perforating the filter. They are then dissolved with as little 
acetic acid as possible, diluted to 50 c.c. with distilled water, and 
titrated with the uranium solution as described. The difference 
between the total amount of P 2 5 and the amount thus obtained 
indicates the quantity of alkaline phosphates present. 

Removal of the Phosphates from the Urine. — Whenever it is 
necessary to remove the phosphates from the urine in the course of 
an analysis, as is frequently the case, the urine is rendered alkaline 
by the addition of the hydrate of an alkaline earth and precipitated 
with a soluble calcium or barium salt. They may also be precipi- 
tated by means of neutral or basic lead acetate, in which case the 
excess of lead is removed by means of hydrogen sulphide or dilute 
sulphuric acid. 

The Sulphates. 

The sulphuric acid found in the urine is derived essentially from 
the albuminous material which is constantly broken down in the 
body, a very small portion only of the inorganic sulphates being 



CHEMISTRY OF THE URINE. 405 

referable to the mineral constituents of the food. As was pointed 
out in the chapter on Reaction, sulphuric acid is constantly produced 
in the body, and, coming into contact with the so-called neutral 
phosphates present in almost all the tissues, transforms these into 
acid phosphates, according to the equation 

2Na 2 HP0 4 + H 2 S0 4 = 2NaH 2 P0 4 + Na 2 S0 4 , 

both appearing in the urine. The alkaline carbonates, which are 
derived from the organic salts ingested by a process of oxidation, 
are also attacked by the sulphuric acid. 

As the amount of food ingested is gradually diminished a point is 
reached when the body most tenaciously holds any alkaline salts that 
may still be present. A new source for the neutralization of the 
acid is then found in the ammonia, which would otherwise have 
been transformed into urea. 

While the greater portion of the sulphuric acid excreted in the 
urine is found in the form of mineral sulphates, about one-tenth of 
the total amount may be shown to be in combination with aromatic 
substances belonging to the oxy-group ; most important among these 
are the salts of phenol, indoxyl, and skatoxyl. 

Indoxyl and skatoxyl, as will be shown later, are derived from 
indol and skatol, which, together with phenol, are formed during 
the process of intestinal putrefaction. Their amount increases and 
decreases with the degree of putrefaction, and hence serves as an 
index of its intensity. 

The mineral sulphates have been termed preformed sulphates in 
contradistinction to the others, which are known as conjugate or 
ethereal sulphates. In the following pages the former will be desig- 
nated by the letter A, the conjugate sulphates by the letter B y and 
the total sulphates as A + B. 

The amount of A + B excreted in the twenty-four hours by a 
normal individual varies between 2 and 3 grammes, the ratio of A 
to B being as 10 : l. 1 

From what has been said, it is apparent that the elimination of 
sulphates is largely dependent upon the degree of albuminous decom- 
position taking place in the tissues and fluids of the body, and hence 
to a certain extent upon the quantity of proteid material ingested, 
the mineral sulphates occurring in such small amount in the food as 
scarcely to affect the quantity excreted. Secondarily, the degree of 
intestinal putrefaction plays a role. The excretion of A -f B is thus 
increased by a diet rich in animal proteids ; the time after a meal, 
however, at which such an increase can be demonstrated varies 
greatly, depending essentially upon the time necessary for digestion. 
AVith a vegetable diet, on the other hand, the total sulphates will be 
found diminished. During starvation A^B is, of course, also 

1 v. d. Velden, Virchow's Archiv, vol. vii. p. 343. 



406 THE URINE. 

diminished, this diminution affecting A especially ; but in some 
cases B may be considerably increased. 1 

An increase in the elimination of the total sulphates is observed, 
as would be anticipated, in all cases in which an increased tissue- 
destruction is taking place, as in the acute febrile diseases. It must 
be remembered, however, that the quantity excreted is then not 
always greater than during convalescence, the diet remaining the 
same. Here, as elsewhere, in urinary studies, it is necessary to dis- 
tinguish between a relative increase and an absolute decrease. In 
pneumonia and acute myelitis the highest figures have been observed, 
the increased elimination during the febrile period being especially 
marked : 2 

Fever diet. Full diet. 



Fever. No fever. No fever. 

Pneumonia 3.51 gm. 1.47 gm. 2.25 gm. 

Acute myelitis 2.62 gm. 1.52 gm. 2.33 gm. 

During convalescence the excretioU of the sulphates is diminished, 
a retention analogous to that of the chlorides and phosphates taking 
place. In contradistinction to the latter salts, it is in all probability 
not the mineral matter proper that is demanded by the body, but the 
sulphur-containing albuminous material. 

A considerable elimination of A + B has also been observed in 
leukaemia, in which an average of 2.46 grammes is excreted, as com- 
pared with 1.51 grammes by a healthy individual receiving the same 
amount and kind of food. In one case of acute leukaemia 5.8 
grammes were eliminated on the day preceding death. 3 

In diabetes mellitus, diabetes insipidus, oesophageal carcinoma, 
progressive muscular atrophy, pseudohypertrophic paralysis, and 
eczema an increased elimination has likewise been observed, while 
in chronic renal diseases a diminished excretion is the rule. 

A study of the elimination of the conjugate sulphates and of the 
relation existing between A and B in disease is still more important 
than that of the total sulphates ; but in both cases the data available 
are scanty, and further observations are urgently needed, v. Noor- 
den regards the elimination of more than 0.3 gramme of conjungate 
sulphates, in the twenty-four hours, as excessive, the patient being 
on an ordinary mixed diet. 

The conjugate sulphates, as would be expected, are increased in 
all cases of increased intestinal putrefaction. In coprostasis the 
result of carcinoma the ratio of the preformed to the conjugate sul- 
phates, normally 10, may diminish enormously. In one case, reported 
by Kast and Baas, it fell to 2, but rose to 7 and 8, and finally to 9.5 

1 Clare, Inaug. Diss., Dorpat, 1854. 

2 P. Furbringer, Virchow's Archiv, vol. lxxiii. p. 39. 

3 Ebstein, Deutsch. Arch. f. klin. Med., vol. xliv. p. 346. 



CHEMISTRY OF THE URINE. 407 

and 1 5 after an artificial anus had been established. I have myself 
observed a drop to 1.5 in a case of volvulus of ten days' standing. 
Biernacki 2 found an increase in the elimination of conjugate sulphates 
amounting to from 0.15 to 0.5 gramme pro die in cases of chronic 
parenchymatous nephritis, going hand in hand apparently with a 
decrease in the secretion of hydrochloric acid by the stomach ; the 
normal amount, according to his observations, varies from 0.1973 to 
0.2227 gramme. In one case B fell from 0.4382 to 0.1505 during 
the administration of hydrochloric acid, to increase again to 0.4127 
upon its discontinuance. 

In accord with these observations are those of Wasbutzki and 
Kast. 3 The former found an increased elimination of B in cases of 
intense bacterial fermentation taking place in the stomach, while 
hydrochloric acid was either totally absent or present in greatly 
diminished amount. A diminished elimination was observed in cases 
of intense torular fermentation, hyperchlorhydria existing at the 
same time. In the absence of hydrochloric acid a normal or even 
a slightly diminished amount was observed in cases of intense acid 
fermentation, lactic acid and butyric acid being present in large 
quantities. By neutralizing the gastric juice with large doses of 
sodium bicarbonate Kast was able to bring about a marked increase 
in the elimination of B, the ratio A : B having fallen from 10.3- 
16.1 to 2.9-6.1. 

Personal observations have led me to the same conclusion, so that 
the following rules may be formulated : 4 

1. A diminution in the secretion of hydrochloric acid is accom- 
panied by an increased degree of intestinal putrefaction. 

2. An increase in the secretion of hydrochloric acid is usually 
accompanied by a decrease in the degree of intestinal putrefaction. 

3. The degree of intestinal putrefaction may be measured directly 
by the elimination of the conjugate sulphates. 

(See also the chapter on the Aromatic Bodies.) 

In obstructive jaundice the excretion of B is likewise increased ; 
it returns to the normal as soon as the permeability of the biliary 
passages has again become established. The total sulphates were 
found in diminished amount in cases of non-obstructive jaundice. 
In Bohm's 5 cases of catarrhal jaundice the excretion of conjugate 
sulphates varied between 0.4 and 0.7. gramme. Of interest in this 
connection are the observations of Muller 6 who notes the elimination 
of 0.29, 0.24, and 0.28 gramme of conjugate sulphates on three 
consecutive days in a case of total obstruction of the biliary duct in 

1 Kast u. Baas, Munch, med. Woch., 1888. 

2 Biernacki, Deutsch. Arch. f. klin. Med., vol. lxix. 

3 Kast, Festsch. z. Eroffnung d. neuen allgem. Krankenhauses, Hamburg, 1889. 
Wasbutzki, Arch. f. exper. Path. u. Pbarmakol., vol. xxvi. 

4 C. E. Simon, Am. Jour. Med. Sci., 1895, vol. ex. 

a A. Bohm, Deutsch. Arch. f. klin. Med., 1901, vol. lxxi. p. 73. 
6 Miiller, Zeit. f. klin. Med., 1887, vol. xii. 



408 THE URINE. 

consequence of a stone. The patient during this period was on a milk 
diet, and there can be little doubt that the low values are here refer- 
able to the germicidal properties of the milk. On a meat diet the 
same patient passed 0.48 and 0.51 gramme. Other observers have 
obtained less constant results in their cases of catarrhal jaundice. 
In cases of hepatic cirrhosis and malignant disease of the liver 
Eiger 1 and Hopadze 2 found increased amounts of conjugate sul- 
phates. 

In cases of diarrhoea A + B y as well as B, is diminished, while 
A : B is increased. 

Of drugs, large doses of morphin, potassium bromide, sodium 
salicylate, and antifebrin appear to cause an increased elimination of 
the total sulphates, while alcohol slightly diminishes the excretion. 

Most important are the observations which have established a 
diminished excretion of the conjugate sulphates following inges- 
tion of the terpen es and camphor, Karlsbad and Marienbad water, 
which latter two, however, at first cause an increase. Kefir, in doses 
of from 1 to 1.5 liters pro die, has proved a most excellent remedy 
with which to combat intestinal putrefaction. Injections of tannic 
acid and of a saturated solution of boric acid apparently produce little 
effect unless the dose is so large as to cause symptoms of poisoning. 

Tests for the Sulphates in the Urine. — The detection of the 
preformed and the combined sulphates iu the urine depends upon the 
fact that the sulphates of the alkalies are precipitated by barium 
chloride as insoluble barium sulphate, according to the equation : 

K 2 S0 4 + BaCl 2 = BaS0 4 + 2KC1. 

In the urine the addition of barium chloride at the same time causes 
a precipitation of the phosphates. These must be kept in solution 
by the addition of an acid, acetic acid being employed for this pur- 
pose whenever the presence of the preformed sulphates is to be 
demonstrated ; hydrochloric acid is inadmissible, as it would cause 
decomposition of the conjugate sulphates and set free the sulphuric 
acid thus held. 

To test for the preformed sulphates, a few cubic centimeters of urine 
strongly acidified with acetic acid are treated with a few drops of a 
solution of barium chloride, when in their presence a cloud or a 
white precipitate of barium sulphate will occur. 

To test for the conjugate sulphates, 25 c.c. of urine are treated 
with about the same volume of an alkaline barium chloride mixture 
(2 volumes of a solution of barium hydrate and 1 volume of a solu- 
tion of barium chloride, both saturated at ordinary temperatures) 
and filtered after a few minutes, the preformed sulphates as well as 

1 Eiger, Inaug. Diss., St. Petersburg, 1893. 

2 Hopadze, Wratsch, 1893, Nos. 48-50. 

3 Ziilzer, Uuters. iiber d. Semiol. d. Harns, Berlin, 1884. 



CHEMISTRY OF THE URINE. 



409 



Fig. 92. 



Fig. 93. 





A Gooch filter. 



the phosphates being thus removed. The filtrate is then strongly- 
acidified with hydrochloric acid and boiled ; the occurrence of a pre- 
cipitate is referable to conjugate sulphates. 

Quantitative Estimation of the Sulphates. 1 — The principle of 
the method employed is the 
same as that just described, 
the preformed sulphates 
contained in the urine 
forming an insoluble pre- 
cipitate of barium sulphate 
when treated directly with 
barium chloride, while the 
combined sulphates do so 
only after having been de- 
composed with strong hy- 
drochloric acid with the 
application of heat. In or- 
der to estimate the amount 
of preformed and conjugate sulphates, it is best to 
determine the total sulphates in one portion, and 
the combined sulphates in another, the difference 
between the two giving the preformed sulphates. 

Quantitative Estimation of the Total Sulphates. — 
One hundred c.c. of clear filtered urine are treated 
with 8 c.c. of hydrochloric acid (specific gravity 
1.12) and heated to the boiling-point, when 20 c.c. 
of a hot saturated solution of barium chloride are 
added. The mixture is kept on a water-bath 
until the barium sulphate has settled down and 
the supernatant fluid appears clear ; this usually 
requires about a half hour. The precipitate is now 
filtered off through a Schleicher and Schull filter, 
or a Gooch filter (Fig. 92), provided with a close- 
fitting plug of asbestos, the whole having been pre- 
viously dried and weighed. Care should be taken never to allow 
the filter to run dry, and small amounts of hot water must be added 
to the last cubic centimeters remaining, the final traces being placed 
upon the filter with the aid of a rubber-tipped glass rod. The pre- 
cipitate is washed with boiling water until a specimen of the wash- 
ings is no longer rendered cloudy, even on standing a few minutes 
after the addition of a drop of dilute sulphuric acid. Gum-like 
substances, as well as pigments, are removed by washing with hot 
alcohol (70 per cent.), and then filling the filter two or three times 
with ether. A suction apparatus is very convenient, but not neces- 

1 E. Salkowski, Zeit. f. physiol. Chem., 1886, vol. x. p. 346; and Virchow's Archiv, 
1888, vol. lxxix. p. 551. 




A suction- funnel. 



410 THE URINE. 

sary ; a simple glass tube, bent upon itself, will answer the purpose 
(Fig. 93). 

If a paper filter has been used, it is placed in a weighed platinum 
or porcelain crucible and ignited. The ash is then heated, at first 
moderately, and almost completely covered with the lid. It is then 
heated, only half covered, for from five to seven minutes, until the 
contents of the crucible are white. The crucible, when cooled, is 
placed in a desiccator and weighed, the difference between the first 
and the second weighing giving the weight of the barium sulphate 
obtained from 100 c.c. of urine. 

A reduction of some of the sulphate usually takes place during 
the process of combustion, owing to the presence of organic matter, 
so that the weight obtained is actually too low. This error may be 
corrected in the following manner : the barium sulphate is washed 
into a small beaker with a small amount of water and titrated with 
a one-tenth normal solution of sulphuric acid, using phenolphthalein 
as an indicator. Each cubic centimeter of the one-tenth normal 
solution corresponds to 0.004 gramme of barium sulphate. The 
actual amount of sulphates contained in 100 c.c. of urine is ascer- 
tained by adding the figure thus found to that obtained by weighing 
(see below). 

Instead of correcting as just described, the ash may be moistened 
with a few drops of a dilute solution of sulphuric acid ; then when 
heat is applied again any sulphide that may have formed is trans- 
formed into the sulphate. 

Quantitative Estimation of the Conjugate Sulphates. — One hun- 
dred c.c. of clear filtered urine are mixed with 100 c.c. of an alka- 
line solution of barium chloride (see above), the mixture being 
thoroughly stirred. After a few minutes it is filtered through a 
dry filter into a dry graduate to the 100 c.c. mark. This portion, 
corresponding to 50 c.c. of urine, is now strongly acidified with 
dilute hydrochloric acid and brought to the boiling-point. It is 
kept upon a boiling water-bath until the barium sulphate has settled 
and the supernatant fluid is clear. The precipitate is filtered off, 
washed, dried, and weighed, as described above. The weight thus 
obtained, multiplied by 2 and deducted from the amount found 
according to the first method, indicates the amount referable to the 
preformed sulphates. The molecular weight of BaS0 4 being 232.82, 
that of S0 3 79.86, of H 2 S0 4 97.82, and of S 32, the figure express- 
ing the amount of H 2 S0 4 , SO s , or S, corresponding to 1 gramme of 
BaS0 4 , is found according to the following equations : 

232.82 : 79.86 : : 1 : x ; and x = 0.34301. .-. 1 gramme of BaS0 4 
=? 0.34301 gramme of SO s . 

232.82 : 97.82 : : 1 : x ; and x = 0.42015. .-. 1 gramme of BaSo 4 
— 0.42015 gramme of H 2 S0 4 . 

232.82 : 32 : : 1 : x ; and x = 0.13744. .-. 1 gramme of BaS0 4 = 
0.13744 gramme of S. 



CHEMISTRY OF THE URINE. 411 

To calculate results, it is only necessary to multiply the weight 
of the BaSO, by 0.34301, 0.42015, or 0.13744, in order to ascertain 
the amount of sulphuric acid contained in 50 c.c. of urine, in terms 
of S0 3 , H 2 S0 4 , or S, respectively. 

Neutral Sulphur. 

While the greater portion of the sulphur of the body is eliminated 
in an oxidized form, small amounts of non-oxidized sulphur bodies 
are likewise found in every urine. They are collectively spoken of 
as the neutral sulphur of the urine, and under normal conditions 
constitute from 12 to 15 per cent, of the total sulphur. The rela- 
tion existing between the oxidized and the neutral form is, however, 
inconstant, and varies with the character of the diet, the degree of 
the proteid metabolism, etc. 

Of the nature of the neutral sulphur bodies which occur in 
normal urine, comparatively little is known. At the present time 
we are acquainted with only two substances belonging to this order, 
viz., certain sulphocyanides and cy stein, or a body which is closely 
related to it. The greater portion of the sulphocyanides is undoubt- 
edly derived from the saliva that has been swallowed and absorbed, 
while a smaller amount may be referable to the trace which is said 
to be present in normal, uncontaminated gastric juice. The amount 
of sulphur which is present in this form represents about one-third 
of the total quantity of the neutral sulphur. Cy stein, probably 
is an intermediary product of the normal metabolism of proteid 
material. Under normal conditions, however, the greater portion 
is oxidized to sulphuric acid, and traces only escape to be eliminated 
as such. 

Whether or not tauro-carbaminic acid, which is a derivative of 
taurin, is a constant constituent of the urine, remains an open question, 
but is very probable. We know, as a matter of fact, that the amount 
of neutral sulphur undergoes a distinct diminution in animals when 
the bile is prevented from entering the intestinal canal by establish- 
ing an external fistula. Under pathological conditions a correspond- 
ing increase is observed in cases of biliary obstruction, and the 
amount of neutral sulphur may then reach 40 per cent, of the total 
sulphur. 

Thiosulphates, which are normally present in the urine of dogs 
and cats, do not occur in human urine under normal conditions. 
That they may be present in disease has been shown by Strumpell, 
who found them in a case of typhoid fever. Further observations, 
hoAvever, are wanting. 

Another sulphur body belonging to this class, which Abel dis- 
covered in the urine of dogs, and which appears to be identical with 
ethyl sulphide, has not as yet been found in the urine of man. 



412 THE VRINR 

The greatest increase in the amount of the neutral sulphur is 
observed under certain conditions associated with the appearance 
of cystin. Normally this is not present in the urine, while traces 
of cystein, or a closely related substance, as I have already stated, 
are found. Cystin is of albuminous origin, and as a matter of fact 
it has been ascertained that all of the loosely combined sulphur 
and even a portion of the firmly combined form exists in the albu- 
mins in the form of the cystin complex. According to Baumann 
and v. Udranszky, its appearance in the urine is closely con- 
nected with the formation of certain diamins, viz., cadaverin, 
putrescin, and a third diamin which is probably identical with saprin 
or neuridin. As these diamins were hitherto supposed to result 
only from the action of certain specific bacteria upon albuminous 
material, cystinuria was regarded as evidence of a definite infectious 
process. It is to be noted, however, that cystin itself does not 
occur in the feces, and that diaminuria does not necessarily accom- 
pany the cystinuria. As the result of personal observations I have 
been led to the conclusion that a causal connection does not exist 
between the two conditions, and that the diamins in question can 
be produced in the body-tissues directly without the intervention of 
micro-organisms. I regard cystinuria essentially as a metabolic 
anomaly, the result of a specific deficiency of the oxidation-processes 
taking place in the body. The condition may be temporary, but as 
a rule it is permanent. It may occur among several members of 
the same family, but it is noteworthy that no case has as yet been 
reported in which a parent and child both were cystin uric. Con- 
sanguinity among parents, which is not infrequently observed in 
cases of alkaptonuria, is the exception in cystinuria. 

The amount of neutral sulphur which may be met with in cystin- 
uria is subject to wide variation, but not infrequently exceeds 30 
per cent, of the total sulphur. As a general rule, the amount of 
cystin eliminated in the twenty-four hours is less than 0.5 gramme. 
At times, however, larger quantities are found, and on one occasion 
I obtained more than 1 gramme. Clinically it is of interest in so far 
as its continued production may give rise to the formation of calculi. 

Unless cystin occurs as a deposit, its presence will scarcely be 
suspected. The substance, however, may occur also in solution, 
and it not infrequently happens that attention, is first drawn toward 
its existence in this state owing to the marked odor of hydrogen 
sulphide, which such urines develop on standing (see Hydrothion- 
uria). If acetic acid is then added in excess, the characteristic 
hexagonal plates may crystallize out. The same result is obtained 
also by allowing the urine to undergo ammoniacal decomposition, 
as cystin is insoluble in solutions of ammonium carbonate. 

Structurally cystin is the disulphide of cystein which latter is 



CHEMISTRY OF THE URINE. 413 

/2-amido-thiolactic acid. On reduction it is transformed into cystein 
according to the equation : 

CxIo'S S.CH 2 CHo-Sil 

I I I 

CH.NH 2 CH.NH 2 + 2H = 2CH.NH 2 

II- I 

COOH COOH COOH 

Cystin crystallizes in hexagonal plates which are quite characteristic, 
and not likely to be confounded with other crystalline elements that 
may be present in urinary sediments. If doubt should arise, their 
solubility in ammonia and hydrochloric acid, and their insolubility 
in acetic acid, water, alcohol, and ether, will lead to their identifi- 
cation. 

The quantitative estimation of cystin is rather unsatisfactory, as 
no method is known which yields reliable results. On the whole, it 
is perhaps best to determine the neutral sulphur, and to refer the 
increase beyond its normal value to the presence of cystin. 

Quantitative Estimation of the Neutral Sulphur in the Urine. — In 
100 c.c. of urine the oxidized sulphur, viz., the mineral and the 
conjugate sulphates, are estimated as described on page 409. In 
the second portion the total sulphur then is determined, the differ- 
ence indicating the amount of the neutral sulphur. 

To determine the total amount of sulphur, 100 c.c. of urine are 
treated with 12 grammes of a mixture of sodium and potassium 
carbonate (11 : 14), and evaporated to dryness in a nickel crucible. 
The residue is fused thoroughly, allowed to cool, and extracted with 
hot water. The carbonaceous residue is filtered off and the filtrate and 
washings are treated with a few crystals of potassium permanganate. 
After heating for about fifteen minutes (more potassium permanga- 
nate should be added if during this time the solution becomes de- 
colorized, when heat is again applied for fifteen minutes), concentrated 
hydrochloric acid is added until the reaction is distinctly acid. This 
solution is then brought to the boiling-point and treated with about 
20 c.c. of a saturated solution of barium chloride. The barium 
sulphate thus formed is then collected and weighed as described on 
page 339. The difference between this result and the first indicates 
the amount of neutral sulphur. 

The total amount of sulphur in the urine is still more conveniently 
determined according to the method of Glaser, as modified by Mod- 
rakowsky : l 1 or 2 grammes of sodium peroxide are placed in a 
nickel dish, and covered with 50 c.c. of urine, added drop by drop. 
The fluid is evaporated to a syrup on a water-bath, and further 
treated with 2-3 grammes of the peroxide, which is added slowly 
while stirring. As soon as the reaction, which at first is quite 
vigorous, has subsided somewhat, the dish is removed from the 
water-bath and heated with a small alcohol lamp. If necessary, 1-3 

1 Modrakowski, Zeit. f. phys. Cheni., 1903, vol. xxxviii. p. 562. 



414 THE URINE. 

grammes more of the peroxide are added. The mass now forms 
brown drops and finally becomes thick ; this ends the reaction. On 
cooling, the fusion is dissolved in hot water ; the solution is filtered 
and feebly acidified with hydrochloric acid. Barium chloride is 
then added and the process continued as described above (page 409). 

Literature. — E. Salkowski, Virchow's Archiv, vol. lxvi. p. 313, and vol. cxxxvii. 
p. 381. Goldmann u. Baumann, " Zur Kenntniss der Schwefelhaltigen Verbindungen 
des Harns," Zeit. f. physiol. Chem., vol. xii. p. 254. E. Salkowski, Virchow's Archiv, 
vol. lviii. p. 461. J. Munk, Ibid., vol. lxix. p. 354; and Deutsch. med. Woch., 1877, 
No. 46. O. Schmiedeberg, " TJeber das Vorkommen von TJnterschwefliger Saure im 
Harn," Arch. d. Heilk., vol. viii. p. 425. A. Strunipell, Ibid., vol. xvii. p. 390. J. Abel, 
" Ueber das Vorkommen von Ethylsulfid im Hundeharn," etc., Zeit. f. physiol. Chem., 
vol. xx. p. 253. (See also Cystinuria and Hydrothionuria.) C. E. Simon, " Cystinuria 
and its Eelation to Diaminuria." Am. Jour. Med. Sci., 1900, vol. cxix. p. 39. C. E. 
Simon and M. W. Lewis, "Transitory Cystinuria," Ibid., 1902, vol. cxxiii. p. 838. 

Urea. 

Urea is by far the most important nitrogenous constituent of the 
urine, and normally represents from 85 to 86 per cent, of the total 
amount of nitrogen which is eliminated by the kidneys. Chemically, 
it may be regarded as carbamide — i. e., as the amide of carbonic 
acid — and is represented by the formula 

/NH 2 

CO< 
X NH 2 

It is thus a comparatively simple substance, and the question natu- 
rally arises : In what relation does urea stand to the highly complex 
albuminous molecule from which it is derived? Numerous hypoth- 
eses have been offered to explain this problem, but, although we are 
in possession of a number of very suggestive data, a final answer to 
the question cannot be given at the present time. In all likelihood, 
however, the urea may originate from the albumins in different ways. 
During the hydrolytic decomposition of the albumins by acids 
and alkalies bodies are constantly formed which belong to the class 
of amido-acids, and these bodies Schultzen and Nencki have accord- 
ingly regarded as intermediary products in the formation of urea 
within the tissues also. The most important members of this 
group are, leucin, tyrosin, glycocoll, asparaginic acid, and gluta- 
minic acid. They are represented by the formulae : 

/NH 2 
CEL/ — Glycocoll, or amido-acetic acid. 

X COOH 



CIV 
C 6 H 4 < 



>CH.CH 2 .CH.NH 2 .COOH — Leucin, or amido-capronic acid. 

/OH(l) 

x CH 2 .CH(NH 2 ).COOH(4) — Tyrosin, or para - oxy - phenyl - amido - propionic 

acid. 



COOH.CH 2 .CH (NH 2 ). COOH — Asparaginic acid, or amido-succinic acid. 
COOH.CH (NH 2 ). CH 2 .CH 2 .COOH— Glutaminic acid, or amido-glutaric acid. 



CHEMISTRY OF THE URINE. 415 

When introduced into the mammalian organism from without, the 
nitrogen of these bodies appears in the urine, to a large extent at 
least, as urea. An analogous formation from the tissue-albumins 
was hence also supposed to occur, but nothing is known of the 
manner of their transformation in the body into urea. Different 
possibilities suggest themselves. It is thus conceivable that cyanic 
acid — CONH — may be produced as an intermediary product, and 
that urea then results through the interaction of two molecules of 
the substance, in statu nascendi, according to the equation 

/NH, 
CONH -f CONH + H,0 = CO< " + C0 2 

\NH 2 

On the other hand, a transformation of the amido-acids into the 
ammonium salts of the fatty acids standing next in order in the 
downward scale may also be imagined. Ammonium carbonate 
would then result, which, through loss of water, could give rise to 
urea. In the case of glycocoll this transformation could be repre- 
sented by the following equations : 

(1) CH 2 .NH 2 .COOH + 20 = NH..COOH -f C0 2 

Amido-acetic acid. Ammonium 

formate. 

(2) 2NH 4 .COOH + 20 = (N~H 4 ) 2 CO s + H 2 + C0 2 

Ammonium Ammonium 

formate. carbonate. 

/NH 2 

(3) (NH 4 ) 2 C0 3 =C0< + 2H 2 



According to Drechsel, further, the amido-acids are transformed into 
carbonic acid, two molecules of which then unite to form urea, 
carbon dioxide, and water : 

/NH 2 /NH 2 /NH- 

C0< + C0< = C0< " -f C0 2 + H 2 

X)H X 0H \NH 2 

The hypothesis of Schultzen and Nencki regarding the origin of 
urea from amido-acids is supported by the fact that these substances, 
when introduced into the mammalian organism from without, are 
largely transformed into urea during their passage through the body. 
It is known, moreover, that in certain diseases, such as acute yel- 
low atrophy, the urea may disappear from the urine almost entirely, 
its place being taken by leucin and tyrosin. In other conditions, 
however, in which the formation of urea is even more seriously 
impaired, leucin and tyrosin do not appear in the urine, and there 
is a growing tendency among physiologists at the present time to 



416 THE URINE. 

abandon the older view of Schultzen and Nencki, and to explain 
the apparently vicarious elimination of the amido-acids in acute 
yellow atrophy upon a different basis. Leucin and ty rosin are 
normally scarcely ever encountered in the mammalian organism, 
and the opinion now prevails that the greater portion of the nitro- 
gen which is to be eliminated from the body leaves the tissues as 
the ammonium salt of paralactic acid. In the liver this is trans- 
formed into ammonium carbonate, from which urea then results 
synthetically, with the intermediary formation of ammonium car- 
bamate. This transition may be represented by the equations : 



(1) (NH 4 ) 2 C0 3 = CO<^ +H 2 

X).NH 4 

Ammonium Ammonium 

carbonate. carbamate. 

/NH 2 y NH 2 

(2) CO< ' =CO< +H 2 

X O.NH 4 X NH 2 

Ammonium Urea, 

carbamate. 



This hypothesis has much in its favor. We thus find that after 
extirpation of the liver in geese the uric acid, which in birds plays 
the same part as the urea in mammals, disappears, and is largely 
replaced by ammonium lactate. In diseases of the liver, moreover, 
in which an extensive destruction of the parenchyma is taking place, 
as in some cases of acute yellow atrophy, in phosphorus poisoning, 
etc., the elimination of urea is diminished, and in its place a cor- 
responding amount of ammonia in combination with lactic acid is 
found. In dogs in which the liver has been in part excluded from 
the general circulation by the establishment of an Eck-fistula, and 
in which the hepatic artery has at the same time been ligated, the 
elimination of urea is much diminished, while that of ammonia 
increases rapidly so soon as the first symptoms of illness appear in 
the animals. In such cases, owing to the incomplete isolation of 
the organ, ammonium carbamate appears in the urine, instead of 
ammonium lactate. From these observations it is apparent also 
that the synthesis of urea takes place in the liver. This is further 
proved by the fact that on transfusion of isolated livers of dogs 
with blood to which ammonium carbonate or ammonium lactate 
has been added, urea is formed as a result. In other organs of the 
body this synthesis apparently does not occur, but there is evidence 
to show that at least a small amount of urea originates elsewhere 
within the body through processes of hydrolysis. This amount, 
however, is unquestionably slight. That a fraction, moreover, is 
formed from uric acid, and in the last instance from the xanthin- 



CHEMISTRY OF THE URINE. 417 

bases through processes of oxidation, can scarcely be doubted, but 
this transformation apparently also takes place in the liver. 1 

Before going on to a consideration of the quantitative excretion 
of urea in health and disease it will be well to form an idea of its 
ultimate sources. To this end, the theory of Voit 2 should be 
recalled, according to which, albuminous material exists in the body 
in two different forms — i. e., as organized albumin, which is built 
up in the form of the tissues of the body, and as unorganized albu- 
min or circulating albumin, which must be regarded in a manner as 
a reserve, to be used in tissue-repair or to be broken down if not 
used, and to be replaced by the proteids ingested with the next meal. 
It may hence be said that, as in the case of the mineral constituents 
of the urine, the urea is referable on the one hand to the proteids of 
the food, and on the other to the proteids of the body-tissues. It 
is clear then that elimination of urea will continue during starvation. 

It has been stated that 84 to 86.6 per cent, of all the nitrogen 
eliminated in the urine is in the form of urea, the remaining 13.4 
per cent, being excreted as uric acid, hippuric acid, kreatinin, 
xanthin-bases, etc. It might hence be supposed that an accurate 
idea of the degree of tissue-destruction could be formed from a 
quantitative estimation of urea. This, however, is not the case, and 
especially in pathological conditions, as the quantitative relations 
existing between the excretion of urea and the remaining nitrogenous 
constituents are subject to wide variation. In acute yellow atrophy, 
for example, as pointed out above, urea may disappear entirely from 
the urine, the nitrogen being eliminated in the form of other com- 
pounds. Whenever it becomes desirable then to gain an accurate 
insight into the degree of proteid-destruction or proteid-assimilation 
— in other words, into the nitrogenous metabolism — taking place in 
the body, it is necessary to resort to a quantitative determination of 
the total amount of nitrogen excreted by the kidneys ; the quantity 
found is then conveniently expressed in terms of urea. At the 
same time it is customary to express the amount of proteid tissue 
which is destroyed, as muscle-tissue, as this serves as a fair type of 
body-tissue in general. 

As 100 grammes of lean muscle-tissue contain about 3.4 grammes 
of nitrogen, corresponding to 7.286 grammes of urea, 1 gramme of 
the latter is equivalent to 13.72 grammes of muscle-tissue. It is, 

lr The origin of urea : O. Schultzen u. M. ISTencki, Zeit. f. Biol., 1872, vol. viii. p. 124. 
E. Salkowski, Zeit. f. physiol. Chem., 1879, vol. iv. p. 100. v. Knieriem, Zeit. f. Biol., 
1874, vol. x. p. 279. E. Salkowski, Zeit. f. physiol. Cheiu., 1877, vol. i. p. 38. Hoppe- 
Seyler, Physiol. Chem., 1881, p. 810. Drechsel, Jour. f. prakt. Chem.. vol. xv. p. 417, 
vol. xvi. pp. 169 and 180, and vol. xxii. p. 476. M. Hahn, V. Massen, M. ISTencki, and 
J. Pawlow, "La fistula d'Eck," etc., Arch. d. Sci. biol. de St. Petersburg, 1892, vol. i. 

Seat of formation ; W. v. Schroder, Arch. f. exper. Path. u. Pharmakol., 1882, vol. 
xv. p. 364. W. Salomon, Virchow's Archiv, 1884, vol. xcvii. p. 149. Minkowski, 
"Ueber d. Einfluss d. Leberextirpation auf d. Stoffwechsel, " Arch. f. exper. Path, 
u. Pharmakol., 1886, vol. xxi. p. 41, and 1893, vol. xxxi. p. 214. 

2 C. Voit, Physiol, d. alls?. Stoft'wechsels u. d. Ernahrung. Herman's Handbuch d. 
Physiol., 1881, vol. vi. I. p. 301. 
27 



418 THE URINE. 

hence, only necessary to multiply the quantity of urea eliminated in 
the twenty-four hours, corresponding to the total amount of nitrogen 
found, by 13.72, in order to obtain an idea of the extent of albu- 
minous destruction taking place in the body. If accurate results 
are desired, it becomes necessary to determine also the amount of 
nitrogen eliminated in the feces, a knowledge of the quantity in the 
food ingested being, of course, presupposed. 

With all these data given, the nitrogenous metabolism of the body 
can be accurately controlled. 

Example. — A patient eliminates 50 grammes of urea in twenty- 
four hours ; these 50 grammes correspond to 50 X 13.72 — L e., 686 
grammes of lean muscle-tissue ; on the other hand, he ingests an 
amount of nitrogenous material corresponding to only 10 grammes 
of urea, equivalent to 10 X 13.72 — i. <?., 137.2 grammes of muscle- 
tissue. The difference between the amount ingested and that ex- 
creted in this case — i. e., 548.8 grammes — must be referable to the 
destruction of organized albumin. 

When the amount of nitrogen eliminated is equivalent to that in- 
gested, nitrogenous equilibrium is said to exist. A healthy person is 
approximately in this condition. 

It has been pointed out that during starvation urea is still elimi- 
nated from the body, although in diminished amount. The question 
now arises, What happens if at this time an amount of nitrogenous 
food is given which corresponds exactly in amount to that elimi- 
nated? Under such conditions an increased elimination of nitrogen 
takes place, all of the nitrogen ingested, in addition to that resulting 
from a breaking down of body-tissues, being excreted. The amount 
of nitrogen referable to the latter source, however, is somewhat less 
than that eliminated in the total absence of food. Unless starvation 
has been pushed too far, the body accommodates itself to the amount 
of food thus given and nitrogenous equilibrium is restored. If more 
food is allowed, an increased elimination results, which again leads to 
a condition of nitrogenous equilibrium, different levels, so to speak, 
being possible. This is well illustrated by comparing the condition 
of the poorly nourished North German laboring population with 
that of the well-fed merchants, the excretion of urea in the former 
amounting to 17.5 to 33.5 grammes, and in the latter to 30 or even 
40 grammes. 

It is apparent, then, that the elimination of urea, and of nitrogen 
in general, is subject to great variation, depending upon the amount 
ingested and that resulting from tissue-destruction, which in turn is 
influenced largely by the body-weight. A statement in figures 
expressing the daily elimination of urea and of nitrogen would, 
hence, be of very little value, especially in pathological conditions, 
in which the amount of nitrogen ingested is frequently very small. 
The elimination of nitrogen should hence always be compared with 



CHEMISTRY OF THE URINE. 419 

the amount ingested, for which purpose the tables of Konig l will 
be found most convenient. At the same time it must be remem- 
bered that not all tho nitrogen taken into the body as food under- 
goes resorption, and that a variable amount, which in disease may 
be considerable, is eliminated with the feces, so that in accurate work 
this nitrogen also must be taken into account. In order to obviate 
the tedious estimation of nitrogen in the feces, it has been proposed 
to determine the standard amount of urea which should appear in 
the urine of a healthy person under different forms of diet. Such 
experiments, of course, presuppose the control-person to be in a 
condition of nitrogenous equilibrium, which, from what has been 
said above, is readily accomplished, as the human body adapts itself 
with ease to different forms of diet. In private practice, however, 
such a procedure would be difficult, but here approximate results 
can be obtained from a parallel estimation of the chlorides. In 
health the elimination of the chlorides may be placed at about one- 
half of the urea. Whenever the nitrogen resulting from tissue- 
destruction is in excess of that referable to the proteids ingested, this 
relation between the excretion of chlorides and urea will be disturbed, 
as the tissues of the body contain very little sodium chloride. When- 
ever the amount of urea is in excess of the normal amount of chlo- 
rides, as indicated above, an increased tissue-destruction may be in- 
ferred, and vice versa. If, on the other hand, the chlorides are present 
in diminished amount, the conclusion may be drawn that a retention 
of albumins is taking place in the body • this is observed frequently 
during convalescence from acute febrile diseases. 

An increase in the amount of urea, and, as a matter of fact, of all 
the nitrogenous constituents, is observed especially in the acute 
febrile diseases, notwithstanding the diminished ingestion of nitrog- 
enous material, and is due to the greatly increased tissue-destruc- 
tion. 2 An excretion of 50 grammes or more is here frequently 
observed. Formerly it was thought that the fever itself was re- 
sponsible for this increased elimination. But this view became 
untenable when it was shown that the excretion of urea in the 
beginning of a febrile attack is not proportionate to the height 
of the temperature, reaching its highest point only when the fever 
has been continuous for several days. Still larger amounts, more- 
over, may be eliminated when the fever is abating. Similar obser- 
vations have since been made. An increased elimination of nitrogen 
may also be noted in almost every case of ague preceding the onset 
of the fever. The latter, therefore, cannot be the only factor which 
causes the increased excretion of urea, and it has been suggested that 
the cells of the body have lost the power of taking up nitrogen. 
The question, however, whether this is dependent upon the increase 

1 J. Konig, Chemie d. menschlichen Nahrungs u. Genussmittel, Berlin, 1893. 

2 Vogel, Zeit. f. rationelle Med., N. F., vol. iv. p. 362. Huppert, Arch. d. Heilk., vol. 
vii. p. 1. L6bisch,Wien. med. Presse, 1889, vol. xxxix. p. 1521. Huppert u. Eiesellt, Arch, 
d. Heilk., vol. x. p. 329. Bauer u. Kiinstle, Deutsch. Arch. f. klin. Med., vol. xxiv. p. 53. 



420 THE URINE. 

in temperature or the action of certain toxic substances circulating 
in the blood, or upon both, still remains unanswered. 

The large increase in the elimination of nitrogen in febrile dis- 
eases is especially striking in those which end by crisis. This is 
notably the case in pneumonia, in which it may persist for two or 
three days after the occurrence of the crisis. The assumption of an 
underlying insufficiency on the part of the cells furnishes a very sat- 
isfactory explanation for the continued increased elimination of urea. 
An increase beyond the amount eliminated during the febrile stage 
is possibly owing to a retention analogous to that of the mineral 
constituents of the urine. 

Apparently, the only exception to the rule that the amount of 
urea is increased in acute febrile diseases, is acute yellow atro- 
phy, in which the excretion of urea is not only greatly diminished, 
but may cease altogether, its place being taken by other nitrogenous 
bodies, such as ammonium lactate, leucin, and tyrosin. 

Among afebrile diseases in which an increased elimination of urea 
has been noted, may be mentioned the ordinary forms of diabetes 
mellitus, in which the highest figures have been obtained, viz., 150 
grammes or more pro die. This is, in all probability, explained, in 
part at least, by the ingestion of excessive amounts of proteid food 
by such patients, but carefully conducted experiments seem to show 
that a not inconsiderable portion of the urea is directly referable to 
increased tissue-destruction. The cases described by Hirschfeld, 1 
however, which will be considered later on, form an exception to 
this rule. 

An increase is observed also in dyspnoeic conditions, and particu- 
larly in pneumonia, in which it is most marked on the day following 
the greatest difficulty in breathing. These observations, however, 
are not free from objections, as an increase has also been noted in 
conditions of apnoea. 

v. Noorden and Lipman-Wolff have shown that anaemia as such 
is not necessarily associated with a pathological increase in the albu- 
minoid metabolism. But it appears that in pernicious anaemia, at 
least in the bothriocephalus form, there are periods in which an in- 
creased albuminous disintegration does occur. According to Rosen- 
qvist, 2 this is far too extensive to be dependent entirely upon the 
destruction of red corpuscles, but must be associated with changes 
in other nitrogenous tissues of the body. After the expulsion of 
the worms a well-marked nitrogenous retention was observed. 
Similar results were obtained in cases of cryptogenetic pernicious 
anaemia, where periods of marked increase of albuminoid disinte- 
gration alternated sometimes with such of distinct nitrogenous reten- 
tion. Rosenqvist concludes that his observations are strongly in 

1 F. Hirschfeld, " Ueber eine neue klin. Form. d. Diabetes," Zeit. f. klin. Med,, vol. 
xix. pp. 294 and 325. 

2 Bosenqvist, Berlin, klin. Woch., 1901, vol. xxxviii. p. 666, 



CHEMISTRY OF THE URINE. 421 

support of the theory that cryptogenetic pernicious anaemia, like the 
bothriocepkalus form, is also a toxic anaemia. 

A moderate increase has been found in severe cases of leukaemia, 
scurvy, minor chorea, and paralysis agitans. Observations made in 
cases of hystero-epilepsy have given rise to conflicting results. It 
is claimed, on the one hand, that the excretion of urea is diminished 
following convulsive seizures of a hystero-epileptic nature, in contra- 
distinction to an increased elimination following true epileptic attacks. 

In cases of functional albuminuria associated with an increased 
elimination of uric acid or oxalic acid, or of both, as well as in 
numerous cases of gastro-intestinal disease, I have observed an in- 
creased elimination of urea, and believe that in the treatment of these 
diseases a systematic study of the excretion of nitrogen is of funda- 
mental importance. 

Of drugs, an increased elimination is produced by coffee, caffein, 
morphin, codein, ammonium chloride, sodium and potassium chlo- 
rides, lithium carbonate, following the ingestion of large amounts of 
water, etc. The data concerning the action of quinin, salicylic acid, 
cold baths, etc., are conflicting. A large increase has been observed 
in cases of phosphorus poisoning. 

Electricity also appears to exert a marked influence upon the 
excretion of urea, producing an increased elimination. 

The diminished elimination of urea observed in certain diseases of 
the liver, 1 notably in acute yellow atrophy, carcinoma, cirrhosis, and 
even in WeyPs disease, is of especial interest, and is in perfect accord 
with the theory that the liver is the main seat of its production. 

As has been stated, urea may disappear altogether from the urine 
in acute yellow atrophy and also in WeyPs disease, notwithstanding 
the frequently not inconsiderable degree of fever. In cirrhosis, 
hyperaemia of the portal system has been thought to cause the dimi- 
nution, which may be increased further in some cases by the occur- 
rence of ascites. In short, the factors which may be regarded as 
causing a diminished elimination of urea in hepatic diseases may be 
summarized under the following headings : 

1. Destruction of hepatic parenchyma. 

2. Diminished velocity of the flow of blood through the liver. 

3. Insufficient excretion of bile and coincident digestive disturb- 
ances. 

Whenever there is disease affecting that portion of the renal 
parenchyma which is concerned especially in the elimination of urea, 
a diminished amount will, of course, be met with, and carefully con- 
ducted observations upon the excretion of the various urinary 
constituents are here of considerable value from a diagnostic as 
well as a therapeutic standpoint. As the glomeruli of the kid- 

1 Hallerworden, Arch. f. exper. Path. u. Pharmakol., vol. xii. Weintraud, Ibid., 
vol. xxxi. Stadelmarm, Deutseh. Arch. f. kliu. Med., vol. xxxiii. Fawitzki, Ibid., 
vol. xlv. Frankel, Berlin, kliu. Woch., 1878 and 1S92. v. Noorden, Lehrbuch d. Path. 
d. Stoffwechsels, p. 287. 



422 THE URINE. 

neys are mainly concerned in the elimination of water and salts 
from the blood, and as the striated epithelium of the convoluted 
tubules appears to provide for the excretion of urea, the elimination 
of a fair amount of the latter with a diminished elimination of salts, 
the phosphates being of especial interest, as they are derived to 
a large extent from albuminous material, would point more particu- 
larly to glomerular disease. On the other hand, a fair excretion of 
phosphates and a diminished excretion of urea would be indicative 
of tubular disease. Whenever glomeruli and tubuli contprti are 
equally diseased an insufficient elimination of both phosphates and 
urea will be observed. 

While, as a rule, the excretion of urea is greatly increased in 
diabetes mellitus, certain cases, which have been elaborately described 
by Hirschfeld, 1 must be excepted. His researches have established 
beyond a doubt that the resorption of nitrogenous material from the 
intestines may be very much below normal, and with it the elimina- 
tion of urea. Upon these grounds he has advocated the recognition 
of a distinct form of diabetes, which is characterized by a com- 
paratively rapid course, the occurrence of colicky abdominal pains 
before or at the onset of the diabetic symptoms proper, the existence 
of pancreatic lesions in a certain proportion of the cases, a more 
moderate degree of polyuria, etc. 

In mental diseases a diminished excretion of urea has been ob- 
served in melancholia and in the more advanced stages of general 
paresis, while an increase is associated with the increased ingestion 
of food during the first stage of profound dementia. 

Following epileptic, cataleptic, and hysterical seizures, as well as 
in pseudohypertrophic paralysis, a decrease has been noted by some 
observers. 

The diminished excretion observed in Addison's disease has also 
been regarded as of nervous origin. 

All forms of chronic, non-progressive anaemia are associated with 
a decrease, as ate also osteomalacia, impetigo, lepra, chronic rheu- 
matism, etc. In chronic lead poisoning the elimination of urea may 
be greatly diminished. 

Little is known of the influence of drugs in bringing about a 
diminished excretion of urea. 

In conclusion, the relation existing between phosphatic excretion 
and that of nitrogen should be especially noted, for a consideration 
of which, see page 399. 

Properties of Urea. — Urea crystallizes in two forms, viz., in long, 
fine, white needles if rapidly formed, or in long, colorless, quadratic 
rhombic prisms when allowed to crystallize gradually from its solutions. 

At 100° C. it begins to show signs of decomposition; at 130° to 
132° C. it melts ; and when heated still further it is decomposed into 
cyanic acid and ammonia, of which the former is immediately trans- 

1 Loc. cit. 



CHEMISTRY OF THE URINE. 



423 



formed into its polymeric compound, cyanuric acid. The reaction 
which takes place is represented by the equations : 



(1) CO 



/NH, 



CONH-f NH S . 



(2) 



* T H 2 
3CONH =C 3 3 N 3 H3. 



Biuret is formed as an intermediary product during this decom- 
position, 2 molecules of urea yielding 1 molecule of ammonia and 
I molecule of biuret, as represented in the equation 



,NfL 



,NH, 



CO< 



co< 



VNTTT \ 

/SS: - NH + NH, 

CO< 



\ 



CO 



\ 



As this substance, obtained on dissolving the residue remaining after 
all the ammonia has been driven off by careful heating, yields a 
beautiful reddish-violet color when a drop or two of a very dilute 
solution of cupric sulphate is added to its solution alkalinized with 
sodium hydrate, this reaction may be employed as a test in the 
detection of urea (Biuret test). 

Urea is readily soluble in water, fairly so in alcohol, and insoluble 
in anhydrous ether and benzol. The aqueous solution of urea is 
neutral in reaction, but this substance combines with acids, bases, 
and salts to form molecular compounds. 

Of special interest are the compounds of urea with nitric acid, 
oxalic acid, and mercuric nitrate. 

Urea nitrate, C(M 2 H 4 .HN0 3 , crystallizes in two different forms : 
in thin rhombic or six-sided colorless plates, which are frequently 



Fig. 94. 




Urea nitrate crystals. (Krukenburg, after Kuhne.) 

observed arranged like shingles one on top of the other when rapidly 
formed (Fig. 94), while larger and thicker rhombic columns or plates 
are obtained if the process of crystallization is allowed to proceed 



424 



THE URINE. 



more slowly. Urea nitrate is readily soluble in distilled water, while 
in alcohol and in water containing nitric acid it dissolves with 
difficulty. Upon heating, it evaporates without leaving a residue. 

Urea oxalate, CON 2 H 4 .C 2 H 2 4 , crystallizes in rhombic or six-sided 
prisms or plates (Fig. 95), which are less soluble in water than the 
nitrate ; in alcohol and in water containing oxalic acid it is only 
imperfectly soluble. 

Fig. 95. 




Urea oxalate crystals. (Krukenberg, after Kuhne.) 

With mercuric nitrate urea forms three different compounds, accord- 
ing to the concentration of the two solutions, viz., (CON 2 H 4 )Hg 2 (N0 3 ) 4 , 
(CON 2 H 4 ).Hg3(N0 3 ) 6 , and (C(M 2 H 4 ) 2 .Hg(N0 3 ) 2 + 3HgO. The lat- 
ter compound is of special importance, as Liebig's quantitative esti- 
mation of urea was based upon its formation. It results when a 
2 per cent, solution of urea is treated with a dilute solution of mer- 
curic nitrate, the reaction taking place according to the equation 

2CON 2 H 4 + 4Hg (N0 3 ) 2 + 3H 2 = [2(CON 2 H 4 ) 2 Hg(N0 3 ) 2 + 3HgO] + 6HN0 3 . 

Very important is the behavior of urea when treated with a solu- 
tion of sodium hypochlorite or hypobromite, the most usual method 
of estimating urea being based upon this reaction, which may be 
represented by the equation 

CON 2 H 4 + 3NaOBr = 3NaBr + 2N+C0 2 + 2H 2 0. 

In the chapter on Reaction it was pointed out that urine gradually 
undergoes ammoniacal decomposition when exposed to the air, 
and that this process is due to the action of a non-organized ferment ; 
the ammonia is liberated according to the equation 

rrti 

CO/ 2 + H 2 = 2NH 3 + C0 2 . 

X NH 2 

This decomposition may also be effected by heating a watery solu- 
tion of urea in a sealed tube to 100° C 



CHEMISTRY OF THE URINE. 425 

Separation of Urea from the Urine. — Fifty to 100 c.c. of 
urine are evaporated to a syrupy consistence upon a water-bath, and 
extracted with 100 to 150 c.c. of strong alcohol, by rubbing up the 
residue, while still hot, with the alcohol. Upon cooling, the mixture 
is filtered, the alcohol evaporated, and the residue treated with pure 
cold nitric acid. Urea nitrate then separates out either immediately 
or on standing. After twenty-four hours the crystalline mass is 
collected on a muslin filter, well strained, and freed from liquid by 
placing it upon plates of clay. The material is then dissolved in 
hot water, and the solution, if strongly colored, gently warmed with 
animal charcoal and filtered. This solution is neutralized with 
barium carbonate, and rendered alkaline with barium hydrate. The 
urea nitrate is thus decomposed, barium nitrate and urea being 
formed : 

2CON 2 H 4 .HN0 3 +BaC0 3 = 2CON 2 H 4 + Ba(N0 3 ) 2 + H 2 0. 

The barium is now removed by passing a stream of carbon dioxide 
through the solution and filtering off the precipitate. The filtrate 
is evaporated until any barium nitrate still remaining crystallizes 
out. This is removed by decantation, when upon further evapora- 
tion the urea crystallizes out, and may be dried between layers of 
filter-paper and re crystallized from 95 to 98 per cent, alcohol. The 
crystals thus formed may now be subjected to further tests. To this 
end, a few drops of an aqueous solution are added to a few cubic 
centimeters of a sodium hypobromite solution, when in the presence 
of urea bubbles of gas will be given off. With a solution of sodium 
hypochlorite the same result may be obtained, but in this case the 
evolution of gas takes place only upon the application of heat. The 
formation of biuret may also be demonstrated by carefully melting 
a few of the crystals in a test-tube, dissolving the residue when 
cool in a little water, and alkalinizing the solution with a little 
sodium hydrate ; upon the addition of a dilute solution of cupric 
sulphate a beautiful reddish-violet color will develop, owing to the 
presence of biuret. 

The addition of oxalic or nitric acid to a solution of urea will 
give rise to the formation of urea nitrate and oxalate, as described 
above. 

This latter test may very conveniently be made under the micro- 
scope. A drop of the concentrated solution is placed upon a slide, 
covered, and a drop of pure nitric acid added from the side. Crystals 
of urea nitrate will then be seen to separate out, and may be recog- 
nized by their characteristic shingle-like arrangement (see Fig. 94). 

When a urine is very rich in urea the mere addition of nitric acid 
will cause a more or less abundant precipitation of urea nitrate, and 
with this simple test an idea may even be formed of the amount 
present. An appearance of hoar-frost is thus noted when not less 



426 THE URINE. 

than 25 grammes are present in the liter, while the formation of 
spangles of urea nitrate requires the presence of at least 45 grammes, 
and an abundant sediment occurs when 50 grammes or more are 
present. 

Quantitative Estimation of Urea. — Hypobromite Method. — The 
method most commonly used in the clinical laboratory is the one 
based upon the decomposition of urea into carbon dioxide and nitro- 
gen in the presence of sodium hypobromite. The reaction takes 
place according to the equation 

CON 2 H 4 + 3NaOBr = NaBr + C0 2 + 2H 2 + 2N. 

The carbon dioxide thus formed is absorbed by an excess of sodium 
hydrate added to the hypobromite solution, while the nitrogen is set 
free, and can be collected and measured ; the determination of the 
corresponding amount of urea is then a simple matter. 

The only solution that is necessary is one of sodium hypobromite 
containing an excess of sodium hydrate. A 30 per cent, solution 
of the latter should be kept on hand and the sodium hypobromite 
solution prepared when required. To this end, 70 c.c. of the sodium 
hydrate solution are diluted with 180 c.c. of water and treated with 
5 c.c. of bromine in a bottle provided with a ground-glass stopper, 
the mixture being thoroughly shaken until every trace of free 
bromine has disappeared. The sodium hypobromite solution, if 
kept in a perfectly dark and cool place, may be preserved for a week 
or two. The reaction which takes place between the sodium hydrate 
and the bromine may be represented by the equation 

2NaOH + 2Br = NaBr + NaOBr + H 2 0. 

Various forms of apparatus, termed ureometers, have been sug- 
gested for the estimation of urea by this method. One which I 
have found very satisfactory is represented in Fig. 96. It consists 
essentially of a burette, C, with an ascending rubber tube attached 
to the reservoir B, which can be raised or lowered as required for 
the purpose of equalizing the pressure after collection of the gas. 
A descending tube leads to a wide-mouthed bottle, A, which con- 
tains the hypobromite solution. This is closed by a tightly fitting 
rubber stopper, to which a loop of platinum wire is attached carry- 
ing a little bucket made of glass or porcelain ; this can be swung 
from its support by inclining the bottle. 

Method. — The rubber stopper is removed from the bottle A, and 
water poured into B until the system BCA is filled to such an ex- 
tent that the water-level is visible in B above the point where the 
rubber tube is attached. About 25 to 30 c.c. of the hypobromite 
solution are placed in the bottle A, and 2 c.c. of urine in the 
bucket ; this is then attached to the wire loop. The stopper is now 



CHEMISTRY OF THE URINE. 



427 



Fig. 



carefully adjusted and the water in B and C brought to the same 
level, when the first reading is taken. A is then inclined until the 
bucket drops into the liquid below. The nitrogen which is liber- 
ated collects in the burette C ; as 
a consequence the water falls in 
C and rises in B. After twenty 
to thirty minutes the pressure 
in C is equalized by lowering B 
until the water in both tubes 
is at the same level. The sec- 
ond reading is then taken, the 
difference between the two indi- 
cating the volume of nitrogen 
liberated from 2 c.c. of urine at 
the temperature of the water in 
CB, which, as well as the baro- 
metric pressure, should be pre- 
viously noted. 

As the volume of gases is 
greatly influenced by the tem- 
perature, the barometric pressure, 
and the tension of the aqueous 
vapor, it becomes necessary, in 
order that the results reached 
shall be comparable with those 
obtained by other observers, to 
reduce the volume of nitrogen 
actually noted to a certain stand- 
ard. This has been placed at 0° 
C. and 760 mercury millimeters 
pressure, in the absence of moist- 
ure. This correction is made ac- 
cording to the following formula : 

V = y^ ^ , in which V represents the corrected 

760.(1 + 0.00366.*)' ^ 

volume of the gas in terms of c.c, v the volume actually ob- 
served, B the barometric pressure in Hgmm., T the tension of the 
aqueous vapor at the temperature noted, t. The volume of nitrogen 
observed being thus corrected, the calculation of the corresponding 
amount of urea is based upon the following considerations : from 
the formula CON 2 H 4 it is apparent that 2 atoms of nitrogen are 
contained in 1 molecule of urea ; in other words, that 28 parts by 
weight of nitrogen correspond to 60 parts by weight of urea. The 
equivalent of 1 gramme of urea is then found according to the 
equation : 60 : 28 : : 1 : x ; and x = 0.46666. The volume corre- 
sponding to 0.4666 gramme of dry nitrogen at 0° C. and 760 




428 



THE URINE. 



Hgmm. pressure is 372.7 c.c. It has been found, however, that 
only 354.3 c.c. of nitrogen are evolved from 1 gramme of urea at 
best when the hypobromite method is employed. Knowing that 
354.3 c.c. of nitrogen correspond to 1 gramme of urea, the amount 
of urea to which the volume of nitrogen actually observed is refer- 
able would then be found according to the equation 

y 



1 : 354.3 : : x : y; and x 



in which y denotes the number of 



354.3' 

cubic centimeters of nitrogen evolved from 2 c.c. of urine, and x the 

corresponding amount of urea. In 
order to ascertain the percentage- 
amount of urea it is only necessary 
to multiply the figure just obtained 
by 50. 

Precautions : 1 . The urine must be 
free from albumin. 2. It should con- 
tain only about 1 per cent, of urea — 
i. e., not more than 0.025 gramme in 
2 c.c. Whenever a greater amount 
is noted, therefore, the urine is diluted 
to the proper degree, due allowance 
being made in the calculation. 

In ordinary clinical work the 
barometric pressure, as well as the 
tension of the aqueous vapor, may 
be ignored, and in the accompany- 
ing tables the corresponding amount 
of urea may be directly read off at 
the temperatures 5°, 10°, 15°, 20°, 
25°, and 30° C. 

Of other forms of apparatus, the 
ureometers devised by Doremus, 
Green, Marshall, Huffner, and 
Squibb may be mentioned. 

The latest modification of Dore- 
mus 7 apparatus is certainly most 
convenient, and can be highly rec- 
ommended. Its general construction is seen in Fig. 97. A small 
amount of urine is poured into B while the stopcock (C) is closed. 
This is then opened for a moment and again closed, so as to fill its 
lumen. The tube A is washed out with water and filled with the 
hypobromite solution. The tube B is filled with urine, and 1 c.c. 
(or less, if the urine is concentrated) is allowed to mix with the hypo- 
bromite solution in A. After all bubbles of gas have disappeared 
the reading is taken. The degrees marked upon the tube indicate 




Doremus' ureometer. 



CHEMISTRY OF THE URINE. 



429 



Urea. Table foe, a Temperature of 5° C. 





1 


1 
10 


2 
10 


_3_ 
10 


4 
10 


5 
10 


6 
10 


7 
10 


_8_ 
10 


_9_ 

10 


1 


1.32 


1.45 


1.58 


1.71 


1.85 


1.98 


2.11 


2.24 


2.37 


2.51 


2 


2.64 


2.77 


2.90 


3.03 


3.17 


3.30 


3.43 


3.56 


3.69 


3.83 


3 


3.96 


^4.09 


4.22 


4.36 


4.49 


4.62 


4.75 


4.88 


5.02 


5.15 


4 


5.28 


5.41 


5.54 


. 5.68 


5.81 


5.94 


6.07 


6.20 


6.34 


6.47 


5 


6.60 


6.73 


6.87 


7.00 


7.13 


7.26 


7.39 


7.53 


7.66 


7.79 


6 


7.92 


8.05 


8.19 


8.32 


8.45 


8.58 


8.71 


8.85 


8.98 


?.ll 


7 


9.24 


9.38 


9.51 


9.64 


9.77 


9.90 


10.04 


10.17 


10.30 


10.43 


8 


10.56 


10.70 


10.83 


10.96 


11.09 


11.22 


11.36 


11.49 


11.62 


11.75 


9 


11.89 


12.02 


12.15 


12.28 


12.41 


12.55 


12.68 


12.81 


12.94 


13.07 


10 


13.21 


13.34 


13.47 


13.60 


13.73 


13.87 


14.00 


14.13 


14.26 


14.39 


11 


14.53 


14.66 


14.79 


14.92 


15.06 


15.19 


15.32 


15.45 


15.58 


15.72 


12 


15.85 


15.98 


16.11 


16.24 


16.38 


16.51 


16.64 


16.77 


16.90 


17.04 


13 


17.17 


17.30 


.17.43 


17.57 


17.70 


17.83 


17.96 


18.09 


18.23 


18.36 


14 


18.49 


18.62 


18.75 


18.89 


19.02 


19.15 


19.28 


19.41 


19.55 


19.68 


15 


19.81 


19.94 


20.08 


20.21 


20.34 


20.47 


20.60 


20.74 


20.87 


21.00 


16 


21.13 


21.26 


31.40 


21.53 


21.66 


21.79 


21.92 


23.06 


22.19 


22.32 


17 


22.45 


23.59 


22.72 


22.85 


22.98 


23.11 


23.25 


23.38 


23.51 


23.64 


18 


23.77 


23.91 


24.04 


24.17 


24.30 


24.43 


24.57 


24.70 


24.83 


24.96 


19 


25.10 


25.23 


25.36 


25.49 


25.62 


25.76 


25.89 


26.02 


26.15 


26.28 


20 


26.42 


26.55 


26.68 


26.81 


26.94 


27.08 


27.21 


27.34 


27.47 


27.60 


21 


27.74 


27.87 


28.00 


28.13 


28.27 


28.40 


28.55 


28.66 


28.79 


28.93 


22 


29.06 


29.19 


29.32 


29.45 


29.59 


29.72 


29.85 


29.98 


30.11 


30.25 


23 


30.38 


30.51 


30.64 


30.78 


30.91 


31.04 


31.17 


31.30 


31.44 


31.57 


24 


31.70 


31.83 


31.96 


32.10 


32.23 


32.36 


32.49 


32.62 


32.76 


32.89 


25 


33.02 


33.15 


33.29 


33.42 


33.55 


33.68 


33.81 


33.95 


34.08 


34.21 


26 


34.34 


34.47 


34.61 


34.74 


34.87 


35.00 


35.13 


35.27 


35.40 


35.53 


27 


35.66 


35.80 


35.93 


36.06 


36.19 


36.32 


36.46 


36.59 


36.72 


36.85 


28 


36.98 


37.12 


37.25 


37.38 


37.51 


37.64 


37.78 


37.91 


38.04 


38.17 


29 


38.31 


38.44 


38.57 


38.70 


38.83 


38.97 


39.10 


39.28 


39.36 


39.49 


30 


39.63 


39.76 


39.89 


40.02 


40.15 


40.29 


40.42 


40.55 


40.68 


40.81 



Urea. Table for a Temperature of 10° C. 





1 


1 


2 


_3_ 


_4_ 


_5_ 


_6_ 


7 


8 


_9_ 






10 


10 


10 


10 


10 


10 


10 


10 


10 


1 


1.31 


1.43 


1.56 


1.69 


1.82 


1.95 


2.08 


2.21 


2.34 


2.47 


2 


2.60 


2.73 


2.86 


2.99 


3.12 


3.25 


3.38 


3.51 


3.64 


3.77 


3 


3.90 


4.03 


4.16 


4.29 


4.42 


4.55 


4.68 


4.81 


4.94 


5.07 


4 


5.20 


5.33 


5.46 


5.59 


5.72 


5.85 


5.98 


6.11 


6.24 


6.37 


5 


6.50 


6.33 


6.76 


6.89 


7.02 


7.15 


7.28 


7.41 


7.54 


7.67 


6 


7.80 


7.93 


8.06 


8.19 


8.32 


8.45 


8.58 


8.71 


8.84 


8.97 


7 


9.10 


9.23 


9.36 


9.49 


9.62 


9.75 


9.88 


10.01 


10.14 


10.27 


8 


10.40 


10.53 


10.66 


10.79 


10.92 


11.05 


11.18 


11.31 


11.44 


11.57 


9 


11.71 


11.84 


11.97 


12.10 


12.23 


12.36 


12.49 


12.62 


12.75 


12.88 


10 


13.01 


13.14 


13.27 


13.40 


13.53 


13.66 


13.79 


13.92 


14.05 


14.18 


11 


14.30 


14.44 


14.57 


14.70 


14.83 


14.95 


15.09 


15.22 


15.35 


15.48 


12 


15.60 


15.74 


15.87 


16.00 


16.13 


16.26 


16.39 


16.52 


16.65 


16.78 


13 


16.91 


17.04 


17.17 


17.30 


17.43 


17.56 


17.69 


17.82 


17.95 


18.03 


14 


18.21 


18.34 


18.47 


18.60 


18.73 


18.86 


18.99 


19.12 


19.25 


19.38 


15 


19.51 


19.64 


19.77 


19.90 


20.03 


20.16 


20.29 


20.42 


20.55 


20.68 


16 


20.81 


20.94 


21.07 


21.20 


21.33 


21.46 


21.59 


21.72 


21.85 


21.98 


17 


22.11 


22.24 


22.37 


22.50 


22.63 


22.76 


22.89 


23.02 


23.15 


23.28 


18 


23.41 


23.54 


23.67 


23.80 


23.93 


24.06 


- 24.19 


24.32 


24.45 


24.58 


19 


24.72 


24.85 


24.98 


25.11 


25.24 


25.37 


25.50 


25.63 


25.76 


25.89 


20 


26.02 


26.15 


26.28 


26.41 


26.54 


26.67 


26.80 


26.93 


27.06 


27.19 


21 


27.32 


27.45 


27.58 


27.71 


27.84 


27.97 


28.10 


28.23 


28.36 


28.49 


22 


28.62 


28.75 


28.88 


29.01 


29.14 


29.27 


29.40 


29.53 


29.66 


29.79 


23 


29.92 


30.05 


30.18 


30.31 


30.44 


30.57 


30.70 


30.83 


30.96 


31.09 


24 


31.22 


31.35 


31.48 


31.61 


31.74 


31.87 


32.00 


32.13 


32.26 


32.39 


25 


32.52 


32.65 


32.78 


32.91 


33.04 


33.17 


33.30 


33.43 


33.56 


33.69 


26 


33.82 


33.95 


34.08 


34.21 


31.34 


34.47 


34.60 


34.73 


34.86 


34.99 


27 


35.12 


35.25 


35.38 


35.51 


35.64 


35.77 


35.90 


36.03 


36.16 


36.29 


28 


36.42 


36.55 


36.68 


36.81 


36.94 


37.07 


37.20 


37.33 


37.46 


37.59 


29 


37.73 


37.86 


37.99 


38.12 


38.25 


38.38 


38.51 


38.64 


38.77 


38.90 


30 


39.03 


39.16 


39.29 


39.42 


39.55 


39.68 


39.81 


39.94 


40.07 


40.20 



430 



THE URINE. 



Urea. Table for a Temperature of 15° C. 








1 


1 


2 


JL 


-i_ 


5 


_6_ 


r 


_8_ 


_9_ 






10 


10 


10 


10 


10 


10 


To" 


10 


10 


1 


1.28 


1.41 


1.53 


1.66 


1.79 


1.92 


2.04 


2.17 


2.30 


2.43 


2 


2.56 


2.69 


2.81 


2.94 


3.07 


3.20 


3.33 


3.46 


3.58 


3.71 


3 


3.84 


3.97 


4.10 


4.22 


4.35 


4.48 


4.61 


4.74 


4.87 


4.99 


4 


5.12 


5.25 


5.38 


5.50 


5.63 


5.76 


5.89 


6.02 


6.14 


6.27 


5 


6.40 


6.53 


6.60 


6.79 


6.91 


7.04 


7.17 


7.30 


7.43 


7.55 


6 


7.68 


7.81 


7.94 


8.07 


8.19 


8.32 


8.45 


8.58 


8.71 


8.83 


7 


8.96 


9.09 


9.22 


9.35 


9.48 


9.60 


9.73 


9.86 


9.99 


10.12 


8 


10.24 


10.37 


10.50 


10.63 


10.76 


10.88 


11.01 


11.14 


11.27 


11.40 


9 


11.53 


11.65 


11.78 


11.91 


12.04 


12.17 


12.29 


12.42 


12.55 


12.68 


10 


12.81 


12.93 


J 3.06 


13.19 


13.32 


13.45 


13.57 


13.70 


13.83 


13.96 


11 


14.09 


14.22 


14.34 


14.47 


14.60 


14.73 


14.86 


14.98 


15,11 


15.24 


12 


15.37 


15.50 


15.62 


15.75 


15.88 


16.01 


16.14 


16.26 • 


16.39 


16.52 


13 


16.65 


16.78 


16.91 


17.03 


17.16 


17.29 


17.42 


17.55 


17.67 


17.80 


14 


17.93 


18.06 


18.19 


18.31 


18.44 


18.57 


18.70 


18.83 


18.95 


19.08 


15 


19.21 


19.34 


19.47 


19.60 


19.72 


19.85 


19.98 


20.11 


20.24 


20.36 


16 


20.49 


20.62 


20.75 


20.88 


21.00 


21.13 


21.26 


21.39 


21.52 


21.64 


17 


21.77 


21.90 


22.03 


22.16 


22.29 


22.41 


22.54 


22.67 


22.80 


22.93 


18 


23.05 


23.18 


23.31 


23.44 


23.57 


23.69 


23.82 


23.95 


24.08 


24.21 


19 


24.34 


24.46 


24.59 


24.72 


24.85 


24.98 


25.10 


25.23 


25.36 


25.49 


20 


25.62 


25.74 


25.87 


26.00 


26.13 


26.26 


26.38 


26.51 


26.64 


26.77 


21 


26.90 


27.03 


27.15 


27.28 


27.41 


27.54 


27.67 


2779 


27.92 


28.05 


22 


28.18 


28.31 


28.43 


28.56 


28.69 


28.82 


28.95 


29.07 


29.20 


29.33 


23 


29.46 


29.59 


29.72 


29.84 


29.97 


30.10 


30.23 


30.36 


30.48 


30.61 


24 


30.74 


30.87 


31.00 


31.12 


31.25 


31.38 


31.51 


31.64 


31.76 


31.89 


25 


32.02 


32.15 


32.28 


32.41 


32.53 


32.66 


32.79 


32.92 


33.05 


33.17 


26 


33.30 


33.43 


33.56 


33.69 


33.81 


33.94 


34.07 


34.20 


34.33 


34.45 


27 


34.58 


34.71 


34.84 


34.97 


35.10 


35.42 


35.35 


35.48 


35.61 


35.74 


28 


35.86 


35.99 


36.12 


36.25 


36.38 


36.50 


36.63 


36.76 


36.89 


37.02 


29 


37.15 


37.27 


37.40 


37.53 


37.66 


37.79 


37.91 


38.04 


38.17 


38.30 


30 


38.43 


38.55 


38.68 


38.81 


38.94 


39.07 


39.12 


39.32 


39.45 


39.58 



Urea. Table for a Temperature of 20° C. 





1 


JL 

10 


2 

To 


A 


A 


5 
10 


A 


7 
10 


A 


_9_ 
10 


1 


1.26 


1.38 


1.51 


1.63 


1.76 


1.89 


2.01 


2.14 


2.26 


2.39 


2 


2.52 


2.64 


2.77 


2.90 


3.02 


3.16 


3.27 


3.40 


3.53 


3.65 


3 


3.78 


3.91 


4.03 


4.16 


4.28 


4.41 


4.54 


4.66 


4.79 


4.91 


4 


5.04 


5.17 


5.29 


5.42 


5.54 


5.67 


5.80 


5.92 


6.05 


6.17 


5 


6.30 


6.43 


6.55 


6.68 


6.81 


6.93 


7.06 


7.18 


7.31 


7.44 


6 


7.56 


7.69 


7.81 


7.94 


8.07 


8.19 


8.32 


8.44 


8.57 


8.70 


7 


8.82 


8.95 


9.08 


9.20 


9.33 


9.45 


9.58 


9.71 


9.83 


9.96 


8 


10.08 


10.21 


10.34 


10.46 


10.59 


10.71 


10.84 


10.97 


11.09 


11.22 


9 


11.35 


11.47 


11.60 


11.72 


11.85 


11.98 


12.10 


12.23 


12.35 


12.48 


10 


12.61 


12.73 


12.86 


12.98 


13.11 


13.24 


13.36 


13.49 


13.61 


13.74 


11 


13.87 


13.99 


14.12 


14.25 


14.37 


14.50 


14.62 


14.75 


14.88 


15.00 


12 


15.13 


15.25 


15.38 


15.51 


15.63 


15.76 


15.88 


16.01 


16.14 


16.26 


13 


16.39 


16.52 


16.64 


16.77 


16.89 


17.02 


17.15 


17.27 


17.40 


17.52 


14 


17.65 


17.78 


17.90 


18.03 


18.15 


18.28 


18.41 


18.53 


18.66 


18.78 


15 


18.91 


19.04 


19.16 


19.29 


19.42 


19.54 


19.67 


19.79 


19.92 


20.05 


16 


20.17 


20.30 


20.42 


20.55 


20.68 


20.80 


20.93 


21.05 


21.18 


21.31 


17 


21.43 


21.56 


21.69 


21.81 


21.94 


22.06 


22.19 


22.32 


22.44 


22.57 


18 


22.69 


22.82 


22.95 


23.07 


23.20 


23.32 


23.45 


23.53 


23.70 


23.83 


19 


23.96 


24.08 


24.21 


24.33 


24.46 


24.59 


24.71 


24.84 


24.96 


25.09 


20 


25.22 


25.34 


25.47 


25.59 


25.72 


25.85 


25.97 


26.10 


26.22 


26.35 


21 


26.48 


26.60 


26.73 


26.86 


26.98 


27.11 


27.23 


27.36 


27.49 


27.61 


22 


27.74 


27.86 


27.99 


28.12 


28.24 


28.37 


28.49 


28.62 


28.75 


28.87 


23 


29.00 


29.13 


29.25 


29.38 


29.50 


29.63 


29.76 


29.88 


30.01 


30.13 


24 


20.26 


30.39 


30.51 


30.64 


30.76 


30.89 


31.02 


31.14 


31.27 


31.39 


25 


31.52 


31.65 


31.77 


31.90 


32.03 


32.15 


32.28 


32.40 


32.53 


32.66 


26 


32.78 


32.91 


33.03 


33.16 


33.29 


33.41 


33.54 


33.66 


33.79 


33.92 


27 


34.04 


34.17 


34.30 


34.42 


34.55 


34.67 


34.80 


34.93 


35.05 


35.18 


28 


35.30 


35.43 


35.56 


35.68 


35.81 


35.93 


36.06 


36.19 


36.31 


36.44 


29 


36.57 


36.69 


36.82 


36.94 


37.07 


37.20 


37.32 


37.45 


37.57 


37.70 


30 


37.83 


37J5 


38.08 


38.20 


38.33 


38.46 


38.58 


38.71 


38.83 


38.96 



CHEMISTRY OF THE URINE. 



431 



Urea. Table for a Temperature of 25° C. 








■h 


2 
10 


3 
10 


A 


5 
10 


6 
10 


7 
10 


_8_ 
10 


A 


1 


1.24 


1.36 


1.49 


1.61 


1.73 


1.86 


1.98 


2.11 


2.23 


2.35 


2 


2.48 


2.60 


2.73 


2.85 


2.97 


3.10 


3.22 


3.35 


3.47 


3.59 


3 


3.72 


5.84 


3.97 


4.09 


4.22 


4.34 


4.46 


4.59 


4.71 


4.84 


4 


4.96 


5.08 


5.21 


5.33 


5.46 


5.58 


5.70 


5.83 


5.95 


6.08 


5 


6.20 


6.33 


6.45 


6.57 


6.70 


6.82 


6.95 


7.07 


7.19 


7.32 


6 


7.44 


7.57 


7.69 


7.81 


7.94 


8.06 


8.19 


8.31 


8.43 


8.50 


. 7 


8.68 


8.81 


8.93 


9.06 


9.18 


9.30 


9.43 


9.55 


9.68 


9.80 


8 


9.92 


10.05 


10.17 


10.30 


10.42 


10.54 


10.67 


10.79 


10.92 


10.04 


9 


11.17 


li:29 


11.41 


11.54 


11.66 


11.79 


11.91 


12.03 


12.16 


12.28 


10 


12.41 


12.53 


12.65 


12.78 


12.90 


13.03 


13.15 


13.27 


13.40 


13.52 


11 


13.65 


13.77 


13.89 


14.02 


14.14 


14.27 


14.39 


14.52 


14.64 


14.76 


12 


14.89 


15.01 


15.14 


15.26 


15.38 


15.51 


15.63 


15.76 


15.88 


16.00 


13 


16.13 


16.25 


16.38 


16.50 


16.63 


16.75 


16.87 


17.00 


17.12 


17.26 


14 


17.37 


17.49 


17.62 


17.74 


17.87 


17.99 


18.11 


18.24 


18.36 


18.49 


15 


18.61 


18.74 


18.86 


18.98 


19.11 


19.23 


19.36 


19.48 


19.60 


19.73 


16 


19.85 


19.98 


20.10 


20.22 


20.35 


20.47 


20.60 


20.72 


20.84 


20.97 


17 


21.09 


21.22 


21.34 


21.47 


21.59 


21.71 


21.84 


21.96 


22.09 


22.21 


18 


22.33 


22.46 


22.58 


22.71 


22.83 


22.95 


23.08 


23.20 


23.33 


23.45 


19 


23.58 


23.70 


23.82 


23.95 


24.07 


24.20 


24.32 


24.44 


24.57 


24.69 


20 


24.82 


24.94 


25.06 


25.19 


25.31 


25.44 


25.56 


25.68 


25.81 


25.93 


21 


26.06 


26.18 


26.30 


26.43 


26.55 


26.68 


26.80 


26.92 


27.05 


27.17 


22 


27.30 


27.42 


27.55 


27.67 


27.79 


27.92 


28.04 


28.17 


28.29 


28.41 


23 


28.54 


28.66 


28.79 


28.91 


29.04 


29.16 


29.28 


29.41 


29.53 


29.66 


24 


29.78 


29.90 


30.03 


30.15 


30.28 


30.40 


30.52 


30.65 


30.77 


30.90 


25 


31.02 


31.15 


31.27 


31.39 


31.52 


31.64 


31.77 


31.89 


32.01 


32.14 


26 


32.26 


32.39 


32.51 


32.63 


32.76 


32.88 


33.01 


33.13 


33.25 


33.38 


27 


33.50 


33.63 


33.75 


33.88 


34.00 


34.12 


34.25 


34.37 


34.50 


34.62 


28 


34.74 


34.87 


34.99 


35.12 


35.24 


35.36 


35.49 


35.61 


35.74 


35.86 


29 


35.99 


36.11 


36.23 


36.36 


36.48 


36.61 


36.73 


36.85 


36.98 


37.10 


30 


37.23 


37.35 


37.47 


37.60 


37.72 


37.85 


37.97 


38.09 


38.22 ■ 


38.24 



Urea. Table for a Temperature of 30° C. 








JL 


2 


_3__ 


_4_ 


5 


JL 


7 


_8_ 


9 






10 


10 


10 


10 


10 


10 


10 


10 


10 


1 


1.22 


1.34 


1.46 


1.58 


1.71 


1.83 


1.95 


2.07 


2.19 


2.32 


2 


2.44 


2.56 


2.68 


2.80 


2.93 


3.05 


3.17 


3.29 


3.41 


2.54 


3 


3.66 


3.78 


3.90 


4.03 


4.15 


4.77 


4.39 


4.51 


4.64 


4.76 


4 


4.88 


5.00 


5.12 


5.25 


5.37 


5.49 


5.61 


5.73 


5.86 


5.98 


5 


6.10 


6.22 


6.35 


6.47 


6.59 


6.71 


6.83 


6.96 


7.08 


7.20 


6 


7.32 


7.44 


7.57 


7.69 


7.81 


7.93 


8.05 


8.18 


8.30 


8.42 


7 


8.54 


8.67 


8.79 


8.91 


9.03 


9.15 


9.28 


9.40 


9.52 


9.64 


8 


9.76 


9.89 


10.01 


10.13 


10.25 


10.37 


10.50 


10.62 


10.74 


10.86 


9 


10.99 


11.11 


11.23 


11.35 


11.47 


11.60 


11.72 


11.84 


11.96 


12.08 


10 


12.21 


12.33 


12.45 


12.57 


12.69 


12.82 


12.94 


12.06 


13.18 


13.30 


11 


13.43 


13.55 


13.67 


13.79 


13.92 


14.04 


14.16 


14.28 


14.40 


14.53 


12 


14.65 


14.77 


14.89 


15.01 


15.14 


15.26 


15.38 


15.50 


15.62 


15.75 


13 


15.87 


15.99 


16.11 


16.24 


16.36 


16.48 


16.60 


16.72 


16.85 


16.97 


14 


17.09 


17.21 


17.33 


17.46 


17.58 


17.70 


17.82 


17.94 


18.07 


18.19 


15 


18.31 


18.43 


18.56 


18.68 


18.80 


18.92 


19.04 


19.17 


19.29 


19.41 


16 


19.53 


19.65 


19.78 


19.90 


20.02 


20.14 


20.26 


20.39 


20.51 


20.63 


17 


20.75 


20.88 


21.00 


21.12 


21.24 


21.36 


21.49 


21.61 


21.73 


21.85 


18 


21.97 


22.10 


22.22 


22.34 


22.46 


22.58 


22.71 


22.83 


22.95 


23.07 


19 


23.19 


23.32 


23.44 


23.56 


23.68 


23.81 


23.93 


24.05 


24.17 


24.29 


20 


24.42 


24.54 


24.66 


24.78 


24.90 


25.03 


25.15 


25.27 


25.39 


25.51 


21 ' 


25.65 


25.76 


25.88 


26.00 


26.13 


26.25 


26.37 


26.49 


26.61 


26.74 


22 


26.86 


26.98 


27.10 


27.22 


27.35 


27.47 


27.59 


27.71 


27.83 


27.96 


23 


28.08 


28.20 


28.32 


28.45 


28.57 


28.69 


28.81 


28.93 


29.06 


29.18 


24 


29.30 


29.42 


29.54 


29.67 


29.79 


29.91 


30.03 


30.15 


30.28 


30.40 


25 


30.52 


30.64 


30.77 


30.89 


31.01 


31.13 


31.25 


31.38 


31.50 


31.62 


26 


31.74 


31.86 


31.99 


32.11 


32.23 


32.35 


32.47 


32.60 


32.72 


32.84 


27 


32.96 


33.09 


33.21 


33.33 


33.45 


33.57 


33.70 


33.82 


33.94 


34.06 


28 


34.18 


34.31 


34.43 


34.55 


34.67 


34.79 


34.92 


35.04 


35.16 


35.28 


29 


35.41 


35.53 


35.65 


35.77 


35.89 


36.02 


36.14 


36.26 


36.38 


36.50 


30 


36.63 


36.75 


36.87 


36.99 


37.11 


37.24 


37.36 


37.48 


37.60 


37.72 



432 



THE URINE. 



directly the number of grammes or grains of urea contained in the 
amount of urine employed. 1 

Green's apparatus (Fig. 98) consists of a tube, graduated in cubic 
centimeters, which is blown out at the bottom into a wider portion, and 
holds in all about 50 to 60 c.c. The bulb is provided with a side-tube, 
into which a bent funnel-tube can be inserted for the purpose of equal- 
izing the pressure. The side-tube having been detached, the apparatus 
is filled with sodium hypobromite solution, when 2 c.c. of urine (di- 
luted if necessary) are introduced by means of a 
graduated and bent pipette. After all bubbles of Fig. 100. 

gas have disappeared the funnel-tube is inserted into 
the side-opening and filled with hypobromite solution 



Fig. 98. 



Fig. 99. 





Green's ureometer. 



Marshall's ureometer. 



Hurfher's ureometer. 



until the level in both tubes is the same. The volume is then 
noted, corrected, and the corresponding amount of urea calculated 
as described. 

Marshall's apparatus is a conveniently modified form of Green's, 
and is used in the same manner (Fig. 99). 

Huffner's apparatus is excellent (Fig. 100). It consists of a small 
bulb, A, of 5 c.c. capacity, which is separated from a larger bulb, 

C, holding about 100 c.c, by a well-oiled glass stopcock. The 
upper end of C is drawn out to such an extent that the eudiometer 

D, which is about 30 cm. long, 2 cm. wide, and divided into fifths 



1 Instead of employing the solution described on page 355, it is sufficient to fill the 
long arm of the tube with a 40 per cent, solution of caustic soda, and to add 1 c.c. of 
bromine and a sufficient amount of water to fill the bend of the tube, 



CHEMISTRY OF THE URINE. 



433 



of a cubic centimeter, can be passed over it for a short distance. 
The bowl E, fitted over C by means of a cork, serves to hold a 
portion of the hypobromite solution. 

The exact capacity of A and of the lumen of the stopcock must 
be separately determined for each instrument. 

Method. — The bulb A and the lumen of the stopcock are filled 
with urine (which has been diluted, if necessary). The stopcock 
having been closed, C is washed out carefully with distilled water 
and filled with the hypobromite solution until the liquid in the dish 
stands several centimeters above the mouth of C. The eudiometer 
is next filled with the same solution, carefully submerged in the 
liquid contained in the dish, and adjusted over the mouth of C. 
The urine in A is then allowed to mix with the hypobromite solution 
very gradually, by opening the stopcock. After all bubbles of gas 
have disappeared the eudiometer is transferred to a cylinder filled 
with water and thoroughly immersed. After twenty to thirty 
minutes the level of the liquid in the tube and that of the outside 
water are equalized and the reading taken. The temperature of 
the water being likewise noted, the volume of the gas is corrected 
and the corresponding amount of urea calculated. 

Squibb's Method. — This method, like that of Doremus, may be 
highly recommended to the practitioner for its simplicity. The 
apparatus (Fig. 101) consists of two ordinary medicine-bottles, A and 

Fig. 101. 




Squibb's ureometer. 

B. In A the nitrogen is evolved. B is closed by a doubly perforated 
rubber stopper, a straight tube passing through the upper aperture 
and connecting with the bottle A. Another tube, bent downward 
and carrying a clamp, as seen in the figure, leads to a graduated 
cylinder, E. B contains a sufficient amount of water for the bent 
tube to dip into ; 25 to 30 c.c. of the hypobromite solution and a 

28 



434 



THE URINE. 



Fig. 102. 



small tube containing 2 c.c. of urine (diluted if necessary, according 
to the specific gravity) are placed in A, the clamp at E being closed. 
The rubber stopper is now firmly inserted and E opened, when a 
few drops of water, which may be disregarded, will escape. The 
graduated cylinder is then placed beneath the outflow-tube and the 
bottle A inclined. The nitrogen collecting in B displaces its own 
volume of water, which flows out and is collected in E, whence the 
corresponding amount of urea may be calculated or read oif from 
the accompanying tables (pages 429-431). 

It should be mentioned that sodium hypobromite liberates nitro- 
gen not only from urea, but also from the other nitrogenous con- 
stituents of the urine ; the error thus incurred, however, appears 
just to counterbalance the deficit in the amount of nitrogen obtained, 
and corresponds to 1 gramme of urea. 

If greater accuracy is required, the method recently suggested by 
Folin may be employed. 1 

Method of Folin. — This is based upon the following considera- 
tions : At a temperature of about 160° 
C. crystallized magnesium chloride, 
MgCl 2 .6H 2 0, boils in its water of 
crystallization. In such a solution 
urea is quantitatively decomposed into 
ammonia and carbon dioxide within 
one-half hour. If the process is car- 
ried out in acid solution, the ammonia 
can subsequently be distilled off after 
rendering the mixture alkaline, and 
is then titrated. The corresponding 
amount of urea is ascertained by cal- 
culation. At the same time, however, 
the preformed ammonia is obtained, 
and it is hence necessary to eliminate 
this source of error by a separate esti- 
mation of this form. This is con- 
veniently done according to the method 
which has likewise been suggested by 
Folin (see below). 

Method. — Three c.c. of urine care- 
fully measured with a 5 c.c. pipette 
graduated in twentieths are placed in 
an Erlenmeyer flask of 200 c.c. ca- 
pacity, together with 20 grammes of 
magnesium chloride and 2 c.c. of concentrated hydrochloric acid. 
(The magnesium chloride usually contains a small amount of am- 
monia, which must be separately determined.) The flask is closed 
J 0, Folin, geit, f. physiol, Cnem., vol, xxii. p. 504, and vol, xxxvi. p. 333, 




Folin's safety-tube. 



CHEMISTRY OF THE URINE. 435 

t 

with a perforated stopper through which a specially constructed 
safety-tube passes (see Fig. 102). 1 The mixture is now boiled until 
the drops flowing back through the tube produce a hissing sound 
on coming in contact with the solution. After this point has been 
reached, the boiling is continued more moderately for about forty- 
five minutes. Immoderate foaming during this process and the sub- 
sequent distillation is guarded against by adding a small piece of 
paraffin (about the size of 2 coffee beans). The solution while still hot 
is carefully diluted to about 500 c.c. — at first by allowing the water 
to flow drop by drop through the tube ; it is then transferred to a 1000 
c.c. retort, treated with about 7 or 8 c.c. of a 20 per cent, solution 
of sodium hydrate, and the ammonia distilled off into a measured 
amount of a decinormal solution of sulphuric acid. The distillation 
may be interrupted when about 350 c.c. have passed over (viz., after 
about sixty minutes). The distillate is boiled for a moment to 
remove any carbon dioxide which may be present in solution, and 
on cooling is titrated to determine the excess of acid. Each cubic 
centimeter of the decinormal ammonia present in the distillate cor- 
responds to 0.003 gramme, viz., to 0.1 per cent, of urea. 

From this result the amount of preformed ammonia and that 
present in the 20 grammes of magnesium chloride must be deducted. 

Estimation of Nitrogen. — For the purpose of estimating the total 
amount of nitrogen in the urine, the method of Kjeldahl or that of 
Will-Varrentrapp is most conveniently employed. 

Kjeldahl's Method. 1 — Principle. — The organic matter of the urine 
is decomposed by means of sulphuric acid, when all the nitrogen 
which is not present in combination with oxygen is transformed into 
ammonia. After adding sodium hydrate in excess the ammonia is 
then distilled off and received in a known quantity of titrated acid, 
the excess being retitrated with sodium hydrate. In this manner 
the amount of ammonia and the corresponding quantity of nitrogen 
are ascertained, it being remembered that 17 grammes of ammonia 
correspond to 14 grammes of nitrogen. 

Reagents required : 

1. Gunning's mixture. This consists of 15 c.c. of concentrated 
sulphuric acid, 10 grammes of potassium sulphate, and 0.5 gramme 
of cupric sulphate. 

2. A solution of sodium hydrate containing 270 grammes in the 
liter (sp. gr. 1.243). 

3. Pulverized talcum or granulated zinc. 

4. A one-fourth normal solution of sulphuric acid. 

5. A one-fourth normal solution of sodium hydrate. 
Apparatus required (see Fig. 103) : This consists of a retort of 

1 The tube can be obtained from Messrs. Eimer & Amend, of New York. 

2 J. Kjeldahl, " Neue Methode zur Bestimmung des Stickstoffes in organischen 
Korpern," Zeit. f. analyt. Chem., 1883, vol. xxii. p. 366. 



436 



THE URINE. 



about 750 c.c. capacity (A), which is connected with a Kjeldahl 
distilling tube (i?), and through this with a Stadeler condenser (C). 
The ammonia is received in the nitrogen bulb at D. In addition a 
Kjeldahl digesting flask of 200 to 300 c.c. capacity is required. 

Method. — Five or 10 c.c. of urine are placed in the digesting 
flask and treated with Gunning's mixture. To this end, it is best 
to add the sulphuric acid and cupric sulphate first, to heat until sul- 
phuric acid vapors are given off in abundance, and then to add the 
potassium sulphate. The heating is continued until the solution 
becomes entirely clear and almost colorless, the flask being inclined 
at an angle of about 45 degrees. Vigorous ebullition should be 
avoided. 

Upon cooling, the contents of the flask are transferred to the re- 
tort with the aid of a little water, and slowly treated with a moder- 
ate excess of the sodium hydrate solution. As a general rule, 40 



Fig. 103. 




Kjeldahl' s nitrogen apparatus. 

c.c. for each 5 c.c. of sulphuric acid are sufficient. A little pulver- 
ized talcum or a few pieces of granulated zinc are finally added ; the 
retort is connected with the condenser, and the distillation begun. 
This is continued until about two-thirds of the solution have passed 
over. The distillate is received in the nitrogen bulb, which should 



CHEMISTRY OF THE URINE. 



437 



contain a carefully measured quantity of the one-fourth normal 
solution of sulphuric acid. As a general rule, 30 c.c. are sufficient. 
As soon as the distillation is completed the condenser is discon- 
nected, washed out with a small amount of distilled water, and the 
washings added to the distillate. After the addition of a few 
drops of tincture of cochineal or dimethyl-amido-azo-benzol the 
excess of sulphuric acid is retitrated with the one-fourth normal 
solution of sodium hydrate, and the amount found deducted from 
the 30 c.c. used. The titration should be continued until every 
trace of yellow has disappeared and a pure rose color is obtained, 
or, in the case of the dimethyl-amido-azo-benzol, until the last trace 
of red has disappeared and the solution has turned yellow. The 
difference multiplied by 0.0035 will then indicate the amount of 
nitrogen present in the 5 or 10 c.c of urine. The corresponding 
amount of urea is found by multiplying this figure by 20. 

Fig. 104. 




Apparatus for the determination of nitrogen. 

As KjeldahFs method presupposes a thorough knowledge of 
chemical technique, it is well to make at least two parallel estima- 
tions in every case. 

Will-Varrentrapp's Method (as modified by Seegen-Schneider). 1 — 

Principle. — If nitrogenous organic material is heated in intimate 

contact with soda-lime, all the nitrogen is given off in the form of 

ammonia, which is received in a known quantity of acid ; the excess, 

1 Will-Varrentrapp, see Leube-Salkowski, Die Lehre vom Harn. 



438 THE URINE. 

not used in the neutralization of the ammonia, is then determined 
by titration with a solution of sodium hydrate of known strength. 
The amount held by the ammonia is thus ascertained, and from it 
the corresponding amount of nitrogen, it being remembered that 17 
grammes of ammonia correspond to 14 grammes of nitrogen. 
Reagents required : 

1. A quantity of thoroughly fused soda-lime, which, while still 
hot, should be placed in a well-stoppered bottle, where it may be 
kept ready for use for a long time. 

2. A normal solution of sulphuric acid. 

3. A normal solution of sodium hydrate. 

Apparatus required : As is apparent from the accompanying dia- 
gram (Fig. 104), the apparatus consists of a Kjeldahl digesting flask, 
A, of about 100 c.c. capacity, and provided with a neck 10 to 12 cm. 
long ; this is placed in a copper crucet, B, and imbedded in sand. 
The crucet is placed upon a pipe-stem triangle over the flame. The 
neck of the flask is surrounded by a hood of copper or tin plate, C, 
moulded to the flask and reaching not higher than 1.5 cm. below 
the rubber stopper. The latter is doubly perforated, a tube, e 7 
drawn out to a point and closed at the free end, passing through 
one aperture and extending about half-way down the flask, w T hile 
the second passes through the other opening. This second tube, 
c, is connected by means of a short piece of rubber tubing, upon 
which a clamp is placed, with a Will-Varrentrapp apparatus. The 
latter is connected by rubber tubing, upon which a clamp is likewise 
placed, with an aspirating-bottle filled with water and provided with 
a siphon tube. 

Method. — Ten c.c. of the normal sulphuric acid solution are 
placed in the Will-Varrentrapp apparatus, together with a few 
cubic centimeters of a 1 per cent, alcoholic solution of phenol- 
phthalein. A layer of sand about 1 cm. in height is placed in the 
crucet, the clamp a closed, and the flask filled to about one-half its 
height with the soda-lime, when the hood is adjusted and 5 c.c. of 
urine are allowed to flow upon the soda. The rubber stopper is 
quickly adjusted, the rubber tube having been previously connected 
with the Will-Varrentrapp apparatus. The clamp a is now opened, 
the crucet filled with sand, and the heating begun. This is at first 
done carefully with a small flame, but increased gradually until a 
full heat is applied. This is continued for one-half to three-quarters 
of an hour. When drops of moisture are no longer visible in the 
tube c, or when the evolution of gas has entirely ceased, the rubber 
tube of the aspirating-bottle d is slipped on to the Will-Varrentrapp 
apparatus, the clamp b slightly opened, the tip of e broken off, and 
air allowed to pass slowly through the entire system for a quarter 
of an hour, when the flame is extinguished. The Will-Varrentrapp 
apparatus is then detached and its contents titrated with the normal 
solution of sodium hvdrate. 



CHEMISTRY OF THE URINE. 439 

The number of cubic centimeters of the sodium hydrate solution 
employed is deducted from 10 (the number of cubic centimeters of 
the normal sulphuric acid solution, 1 c.c. of the latter being equiva- 
lent to 1 c.c. of the former), the difference giving the number of 
cubic centimeters of the normal sulphuric acid solution neutralized 
by the ammonia evolved from 5 c.c. of urine. This number multi- 
plied by 20 will then represent the number of cubic centimeters 
required to neutralize the ammonia contained in 100 c.c. of urine. 
As 1000 c.c. of the normal solution of sulphuric acid correspond to 
17 grammes of ammonia, or 14 grammes of nitrogen, the number of 
cubic centimeters of the sulphuric acid solution corresponding to 
100 c.c. of urine will be found from the equation : 1000 : 14 : : x : y ; 
and y = 0.01 4 x, in which x represents the number of cubic centi- 
meters required to neutralize the amount of ammonia evolved from 
100 c.c. of urine, and y the corresponding amount of nitrogen — i. e., 
the percentage of nitrogen. 

If the nitrogen is to be calculated in terms of urea, this is done 
according to the equation : 1000 : 30 ( = 14N) : : x : y ; and y = 
0.03 x = percentage of urea, in which x represents, as above, the 
number of cubic centimeters of sulphuric acid neutralized by the 
ammonia, viz., nitrogen, contained in 100 c.c. of urine, and y the 
urea corresponding to this amount. 

Ammonia. 

Every urine contains a small amount of ammonia, which normally 
varies but little, and corresponds to from 4.1 to 4.64 per cent, of the 
total amount of nitrogen, viz., to about 0.7 gramme in the twenty- 
four hours. It is present in combination with the various acids of 
the urine, and in all likelihood represents a small amount of the 
ammonia which has not been transformed into urea, but has been 
utilized to saturate the affinities of a slight excess of acid, formed 
during the nitrogenous metabolism of the body, over the available 
fixed alkalies. In this manner indeed the body is capable of guard- 
ing against the appearance of free acid in the blood, and it is for 
this reason, as I have already pointed out, that free acid cannot 
occur in the urine. This safeguard, however, does not exist in 
the herbivorous animals, in which the fixed alkali only is apparently 
available for the neutralization of acids, and we consequently find 
that whereas in dogs, for example, an acid intoxication occurs only 
after the administration of very large quantities of acid, the herbivora 
rapidly succumb after the ingestion of comparatively small amounts. 

In man an increased elimination of ammonia is observed when- 
ever an increased formation of acids occurs, or whenever a sufficient 
supply of oxygen is not available. In the latter case, no doubt, 
the increased elimination is owing to the fact that in consequence 
of the deficient supply of oxygen the synthetic formation of urea 



440 



THE URINE. 



from ammonium lactate is impeded in the liver. As this organ, 
moreover, is the principal seat of the synthesis of urea, we can 
readily understand that extensive parenchymatous degeneration, 
as in acute yellow atrophy, in phosphorus poisoning, etc., will lead 
to an increased elimination of ammonia. 

In any event, the relative increase of the ammonia is the 
essential factor, while variations in its absolute quantity are of 
secondary importance. Some of the results which have been 
obtained in various diseases are given in the following table : 

Per cent. 

Normal values 4.10- 4.64 

Febrile diseases 5.72- 6.70 

Carcinoma of the liver 6.40-24.50 

Liver abscess (actinomycosis) 10.60 

Circulatory dyspnoea 13.10-32.20 

Respiratory dyspnoea 6.60-14.30 

Abnormally high absolute values are quite constantly observed in 
diabetes, in which an elimination of from 4 to 5 grammes may be 
regarded as common. In one instance 5.94 grammes were excreted 
in twenty-four hours. In a general way the amount of ammonia in 
cases of diabetes gives an idea of the amount of organic acids ; 
but, as Herter has pointed out, we cannot detect moderate quan- 
tities of organic acids in this way (see Oxybutyric Acid). 

Fig. 105. 




Desiccator. 



Very curiously, diminished elimination of ammonia is observed 
in many cases of nephritis so long as symptoms of venous stasis 
do not exist. 

In a case of pernicious aneemia relative amounts, varying between 
3.3 and 5.6 per cent., were obtained during the days immediately 
preceding death. 



CHEMISTRY OF THE URINE. 



441 



Quantitative Estimation. — Schlbsing's Method. — Principle. — A 
carefully measured amount of urine is treated with milk of lime 
and placed under a bell, together with a vessel containing a known 
amount of a normal solution of sulphuric acid. In the course of 
time the ammonia is liberated and absorbed by the acid. This is 
then titrated, and the deficit expressed in terms of ammonia. 

Method. — Twenty-five c.c. of perfectly fresh, filtered urine are 
placed in a flat dish, upon the plate of a desiccator, as shown in 
Fig. 105. Above this is a smaller dish containing 10 c.c. of a nor- 
mal solution of sulphuric acid. The urine is treated with 10 c.c. 
of milk of lime, the bell is carefully adjusted after lubrication 
with tallow, and the apparatus allowed to stand for at least three 
or four days. The excess of acid remaining is then titrated with a 
one-fourth normal solution of sodium hydrate, using as an indicator 
a few drops of a saturated aqueous solution of methyl-orange until 
the red color has turned to yellow. To neutralize the 10 c.c. of the 
acid, 40 c.c. of the one-fourth normal solution are required. The 
difference is referable to the partial neutralization by the ammonia, 
and is expressed in milligrammes. One c.c. of the one-fourth 
normal solution corresponds to 4.25 mgrms. of ammonia. 

Precautions : 1. In every case the urine must be 
perfectly fresh. Decomposition is best guarded Fig. 106. 

against during its collection by adding about 10 to 
20 c.c. of chloroform to the portion first voided. 

2. Urines which are undergoing ammoniacal de- 
composition should not be utilized for examination. 

3. Concentrated or albuminous urines must be 
kept under the bell for from five to eight days, 
new portions of acid being used when in doubt as 
to the complete liberation of the ammonia. 

Owing to a slight deposition of moisture on the 
inner surface of the bell and a consequent reten- 
tion of traces of ammonia in this form, the result- 
ing figures are too low. The error thus incurred, 
however, is insignificant. 

More satisfactory than this older method is the 
following method, which has been suggested by 
Folin : 

Folin's Method. — Ten c.c. of urine are diluted to 
about 450 c.c, treated with a small amount of 
burnt magnesia (0.5 gramme), and boiled for 
forty-five minutes, the distillate being received in 
decinormal sulphuric acid through an absorption- 
tube, such as the one pictured in Fig. 106. This 
consists of a glass tube, a, measuring about 8 mm. in diameter, one 
extremity of which has been blown into the small bulb b. By means 




442 THE URINE. 

of a heated platinum wire five or six holes, each about 1 mm. in 
diameter, are made in the bulb ; c is a rubber stopper which fits into 
the second tube d. This is merely a test-tube (2.5 cm. in diameter) 
which has been cut about 7.5 cm. from the upper end. About 3 cm. 
from the upper margin this tube is provided with six or seven holes 
as in the bulb b. The entire apparatus is directly immersed in the 
decinormal acid and insures the complete absorption of the ammonia 
in one flask, even if this contains only 5—10 c.c. of the acid. The 
ammonia is then determined by titration as above, using alizarin 
red as indicator ; 2 drops of a 1 per cent, solution suffice for 200—300 
c.c. The titration is carried to the red point, not to the violet. As 
a small amount of urea, however, is decomposed during the pro- 
longed ebullition, it is necessary to ascertain separately the quan- 
tity of ammonia which is referable to this source. To this end, 
the retort is opened at the expiration of forty-five minutes, and 
an amount of water added which is approximately equivalent to that 
of the distillate. The distillation is then continued for another period 
of forty-five minutes ; the distillate is received in decinormal sul- 
phuric acid, and the ammonia referable to decomposition of the urea 
estimated as before. The difference between the two results indicates 
the amount of preformed ammonia that was originally present. 

This method is also applicable for the determination of ammonia 
in the blood. 

Literature. — Hallervorden, Arch. f. exper. Path., vol. xii. p. 237. Stadelmann, 
Deutsch. med. Woch., 1889, p. 942. Michaelis, Ibid., 1900, p. 276. O. Folin, Zeit. f. 
physiol. Chem., vol. xxxii. p. 575 ; and Ibid., 1902, vol. xxxvii. p. 161. 

Uric Acid. 

According to our present views, uric acid, in man, is not formed 
during the decomposition of all albuminous substances, as was for- 
merly supposed, but constitutes a specific product of decomposition 
of one class of albumins only, namely, the nucleins. 1 It appears, 
moreover, that the mother-substance of uric acid is confined to the 
nuclear nucleins, viz., to those containing a nucleinic acid radicle ; 
while the paranucleus, in which this is lacking, are without effect 
upon the elimination of uric acid. According to Kossel, 2 four differ- 
ent forms of nucleinic acid exist, viz., adenylic acid, guanylic acid, 
sarcylic acid, and xanthylic acid, and the supposition is that each of 
these contains one base, viz., adenin, guanin, sarcin or hypoxanthin, 
and xanthin. These basic substances are collectively spoken of as 
the xanthin, alloxur, or purin bases. According to Emil Fischer, 1 
they are derived from a hypothetical compound which he terms purin, 
and which he supposes to be constituted as shown in the formula. 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 

2 A. Kossel u. A. Neumann, "Ueber Nukleinsaure u. Thyminsaure," Zeit. f. 
physiol. Chem., vol. xxii. p. 74. 

3 E. Fischer, Ber. d. Deutsch. chem. Ges., 1897, vol. xxx. p. 549. 



CHEMISTRY OF THE URINE. 



443 



(1)N: 

(2)HC 

II 
(3)N- 



(6) 
=CH 



(5)C- 



(4) 



(7) 
-NHn 

-N^ 

(9) 



CH(8). 



By substituting the group NH 2 for the H atom at 6, adenin thus 
results, and is hence also spoken of as 6-aminopurin : 



N: 

H A 

II 



:C.NH 2 

C N^ 



-N 



X 



CH. 



Hypoxanthin, according to this conception, would be 6-oxypurin ; 
xanthin 2, 6-dioxypurin, and guanin 2-amino-6-oxypurin, as shown 
by the structural formula? : 



HN- 

II 
N- 



— CO 

C NH 

— C N^ 

Hypoxanthin. 



CH. 



HN— 

i° 

HN— 



-CO 



-NH 



\ 



-C N 

Xanthin 



^ 



CH. 



NH- 
HN=C 



-CO 



HN- 



-NHn 
-N^ 



CH. 



-C- 

Guanin. 

From the structural formula of purin it is also apparent that still 
other derivatives of this substance may exist, and as a matter of 
fact others are known, viz., mono-methylxanthin or heteroxanthin, 
di-methylxanthin or paraxanthin, tri-methylxanthin, the isomeric 
compounds of paraxanthin, viz., theophyllin and theobromin, and 
others. Their relation to xanthin is shown in the formulae : 



HN- 



CO 



HN 



-CO 

1 

C NH X 

II > 

-C N^ 

Xanthin. 



HN— 

I 
CO 



-CO 

I 
c— 



-N.CH, 



:CH. 



CH 3 .N CO 

I I 



-N.CH, 



CO 

I II ^ 

HN C N=^ 

Paraxanthin. 



:CH. 



HN C N=^ 

Heteroxanthin. 

CH 3 .N CO 

I I 

CO C NHv 

I II ^>CH. 

CH 3 .N C N^^ 

Theophyllin. 




CH. 



CH 3 .N C N= 

Theobromin. 




CH. 



444 THE URINE. 

Two of these bodies, namely, heteroxanthin and paraxanthin, 
have also been found in urine. 

From these basic substances, then, which are found in the nucle- 
inic acid radicle of the nuclear nucleins, uric acid is supposedly 
derived, and there are numerous facts which go to show that this 
supposition is in all likelihood correct. It will thus be observed 
that structurally uric acid is intimately related to the bodies in ques- 
tion, and, like these, contains the purin radicle : 

HN — —CO 

I I 

CO C NH V 

I II >0. 

HN C NH X 

Uric acid. 

It may hence be regarded as 2, 6, 8 tri-oxypurin. Uric acid and 
the xanthin-bases, moreover, qualitatively, all yield the same decom- 
position-products when treated with fuming hydrochloric acid or 
hydriotic acid under high pressure ; only the quantitative relations 
vary, as shown in the equations : 

C 5 H 5 N 5 + 8H 2 = 4NH 3 -f C0 2 + CH 2 .NH 2 .COOH + 2H.COOH. 
Adenin. Glycocoll. Formic acid. 

C 5 H 4 N 4 + 7H 2 = 3NH 3 + C0 2 + CH 2 .NH 2 .COOH + 2H.COOH. 
Hypoxanthin. 

C 5 H 5 N 5 + 7H 2 = 4NH 3 + 2C0 2 + CH 2 .NH 2 .COOH + H.COOH. 

Guanin. 

C 5 H 4 N 4 2 + 6H 2 = 3NH 3 + 2C0 2 + CH 2 .NH 2 .COOH + H.COOH. 

Xanthin. 

. C 5 H 4 N 4 3 + 5H 2 == 3NH 3 + 3C0 2 + CH 2 .NH 2 .COOH. 
Uric acid. 

In accordance with this supposed origin of uric acid we find an 
increased elimination in the urine following the ingestion of all sub- 
stances which contain purin bases either as such or in the form of 
nuclear nucleins. At the same time it must be remembered that 
uric acid may also result from the nucleins of the body -tissues ; and 
we find, as a matter of fact, that during starvation uric acid does 
not disappear from the urine. The principal source of the uric acid 
under such conditions are the nucleins of the leucocytes ; and accord- 
ing to Horbaczewski 1 and others, this source is indeed more impor- 
tant than the nucleins of the food. According to his idea, the latter 
call forth an increased elimination of uric acid in only an indirect 
manner — i. e., by stimulating more strongly than other food-stuffs 
the cell -formation and cell-destruction of the body. However this 

1 J. Horbaczewski, "Beitrage zur Kenntniss der Bildung von Harnsaure," etc., 
Monatshefte fur Chem., 1891, vol. xii. p. 221 ; and Wien. Sitzungsber., vol. c. 



CHEMISTRY OF THE URINE. 445 

may be, there can be no doubt that the amount of uric acid elimi- 
nated in the urine depends, in the first instance, upon the amount of 
nucleins or purin bases as such which are ingested, and upon the 
degree of nuclear destruction which takes place in the body. Other 
factors, however, also enter into consideration. "We thus know that 
the body is capable of transforming a certain amount of uric acid 
into urea. This fact was pointed out long ago by Frerichs and 
Wohler, and has recently again been confirmed. It was found that 
after the ingestion of large amounts of nucleins only a certain por- 
tion of the nuclear nitrogen is eliminated as uric acid, and that this 
amount is extremely variable. Whether individual peculiarities 
have any part in determining this amount is unknown, but is not 
improbable. Oxidation on the part of the body-tissues must also 
be taken into consideration, and it unquestionably varies not only 
in different people, but also in the same individual at different times. 
Then again there is evidence to show that under certain conditions 
uric acid may be formed synthetically in the body. That this is the 
usual mode of formation in birds and reptiles has been conclusively 
shown by Minkowski, 2 who found that after extirpation of the liver 
in geese the greater portion of the urinary nitrogen was eliminated 
in the form of ammonia in combination with lactic acid. In the 
human being very little uric acid is in all likelihood formed in this 
manner under normal conditions, but the possibility of its occur- 
rence, in disease more particularly, should not be overlooked. As 
uric acid, moreover, may in part at least be eliminated in the 
feces, it is clear that the amount which appears in the urine cannot 
be regarded as an accurate index of the degree of nuclear destruc- 
tion or of the amount which is formed in the body-tissues. That 
retention of uric acid can further occur in the body, which may 
or may not be followed by increased elimination, is likewise un- 
doubted. 

According to our present knowledge, uric acid is formed in all the 
organs of the body, including the bone-marrow, the muscles, the 
spleen, the liver, the kidneys, etc. Under pathological conditions it 
may also originate in the joints and tendons. 

Under normal conditions the daily elimination of uric acid varies 
between 0.2 and 1.5 grammes, thus constituting -J^ to y^j- part of 
the total urinary nitrogen. It is largely influenced by the character 
of the diet, the amount of exercise taken, the general health of the 
individual, etc. After the ingestion of large amounts of food rich 
in nuclear nucleins, such as thymus gland, liver, kidneys, and brain, 
a corresponding increase in the amount of uric acid is observed. 
Generally speaking, animal food causes a greater elimination of uric 
acid than vegetable food, and it is supposed that this difference is 

1 Minkowski, "Ueber den Einfluss d. Leberextirpation auf den Stoffwechsel." 
Arch. f. exper. Path. u. Pharmakol., 1886, vol. xxi. p. 41. 



446 THE URINE. 

essentially due to the presence of the extractives of the meat. 1 Of 
special interest is the increase in the elimination of uric acid which 
is observed five hours after the ingestion of a full meal. This in- 
crease, according to Horbaczewski, 2 is associated with the disappear- 
ance of the digestive leucocytosis and consequent leucolysis. 

Some observers have attached much importance to the relation 
existing between the elimination of uric acid and urea, and are in- 
clined to assume the existence of a special uric acid diathesis when 
this relation continuously exceeds the usual standard of 1 : 50 or 
1 : 60. This question is, however, an extremely intricate one, and 
we are scarcely in a position at the present time to speak definitely 
of the significance of such variations. On the one hand, there can 
be no doubt that an unusually high uric acid coefficient may be met 
with in individuals who are apparently in good health, while in others, 
in whom larger amounts of uric acid are eliminated than are usual, 
normal or even subnormal values may be found. The entire ques- 
tion of the uric acid diathesis is in a chaotic condition, and it would 
perhaps be well to speak of such a diathesis only when a distinct 
absolute increase is continuously observed. That numerous symptoms 
of a neurasthenic type are often seen when the uric acid coefficient 
is increased, is a matter of daily observation, but it would be pre- 
mature to regard this symptom as a causative factor of the disease 
in question. 3 Even in gout it can scarcely be said that uric acid has 
been proved the materia peccans, and our knowledge concerning the 
etiology of the disease is still as obscure as when Garrod 4 showed 
that an accumulation of uric acid occurred in the blood of such pa- 
tients. Hitherto it has been supposed that the deposition of urates 
in the joints and periosteum of gouty patients is referable to a 
diminished alkalinity of the blood, and that acute paroxysms result 
whenever an increase in its alkalinity occurrs, leading to a resorp- 
tion of the urates previously deposited and a consequent flooding of 
the system with the material in question. As a matter of fact, a 
considerable diminution in its excretion is observed immediately 
preceding the attack, while during the paroxysm and immediately 
following it a corresponding increase is noted. Numerous investi- 
gations, however, have shown that distinct changes in the alkalinity 
of the blood do not occur in gout, and that an increase in the amount 
of uric acid in the blood is not only observed in this disease, but in 
other diseases as well which are not associated with gouty symptoms. 

1 A. 1 Hermann, " AbMngigkeit der Harnsaureausscheidung von Nahrungs- und Ge- 
nussmitteln," Deutsch. Arch. f. klin. Med., 1888, vol. xliii. p. 273. See also W. Camerer, 
Zeit. f. Biol., N. F., 1896, vol. xv. p. 140. 

2 Horbaczewski, Harnsaureausscheidung u. Leucocytose, Sitzungsber. d. Wiener 
Akad. d. Wissensch., 1891, Abth. 3. See also Lowit, Studien z. Physiol, u. Path. d. 
Blutes, 1892. W. Kiihnau, " Das Verhaltniss d. Harnsaureausscheidung zur Leuco- 
cytose," Zeit. f. klin. Med., vol. xxviii. p. 534. P. F. Eichter, " Ueber Harnsaure- 
ausscheidung und Leucocytose," Ibid., vol. xxvii. p. 290. 

3 C. E. Simon, Am. Jour. Med. Sci., 1899, p. 139 , aim N. Y. Med. Jour., 1895, p. 330. 

4 A. B. Garrod, On the Nature and Treatment of Gout, 1847. 



CHEMISTRY OF THE URINE. 447 

The conclusion is hence justifiable that the presence of uric acid in 
the blood per se cannot be offered as an explanation of the occur- 
rence of a gouty attack. 1 Futcher, 2 who has recently observed a 
number of cases of gout with modern methods, states that he al- 
most invariably found that before the onset of the acute symp- 
toms the uric acid is below and often far below 0.4 gramme. On 
the second or third day after the beginning of the acute symptoms 
the uric acid curve steadily rises, reaching 0.8 to 1.9 gramme 
or even higher values than this. With subsidence of the acute 
symptoms the curve gradually falls below the lower limit of 
the normal, and in the interval between the acute attacks the 
excretion may be only 0.1-0.2 gramme daily. In one very marked 
chronic case Futcher found no uric acid excretion whatever on cer- 
tain days during the interval. The phosphoric acid curve runs a 
course almost parallel to that of the uric acid, which suggests quite 
strongly that even in gout the uric acid is derived from nucleins, 
and is not formed synthetically, as might possibly be imagined. 

The greatest increase in the elimination of uric acid is observed 
in leukaemia, in which amounts of 5 grammes and even more may 
be observed in the twenty-four hours. That the increased elimina- 
tion in this disease is referable to the enormous increase in the 
number of the leucocytes and consequent lencolysis can scarcely be 
doubted. In other diseases which are associated with a high grade 
of leucocytosis, and especially those in which the disease terminates 
by crisis or hastened lysis, such as erysipelas and pneumonia, a con- 
siderable increase is likewise observed, and is referable to the same 
origin. This increase is especially marked immediately after crisis 
has occurred, but it not infrequently precedes this by several hours. 
In the other febrile diseases an absolute increase is less marked and 
inconstant. 

In diabetes a diminished amount of uric acid is usually found. 
Cases may be seen, however, in which, associated with a diminution 
or an entire disappearance of the sugar, a most marked increase occurs, 
amounting in some cases to 3 grammes in the twenty-four hours. To 
this condition the term diabetes alternans has been applied. 

In acute articular rheumatism an increased elimination is observed 
so long as the temperature remains high, while with approaching 
convalescence the amount returns to normal, and may even fall 
below normal. In chronic rhemnatism, on the other hand, no con- 
stant relations have been observed. 

In the ordinary forms of anaemia and chlorosis the amount of 
ivric acid is quite constantly diminished, as also in chronic inter- 

1 B. Laquer, Ueber die Ausscheidungsverhaltnisse der Alloxurkorper. Bergmann, 
1906. (Full literature.) C. von Xoorden, Lehrbuch d. Pathologie d. Stoffwechsels, 
Berlin. 1893. W. Ebstein, "Die Xatur u. Behandluug der Gicht," Verbandl. d. VIII. 
Congr. f. inn. Med., 1889, p. 133. 

2 T. B. Futcher, "The Occurrence of Gout in the United States," Jour. Am. Med, 
Assoc, 1902, vol. xxxix. p. 1016. 



448 



THE URINE. 



stitial nephritis, chronic lead poisoning, progressive muscular atro- 
phy, and pseudohypertrophic paralysis. 

Properties of Uric Acid. — The close relation existing between 
uric acid and the xanthin-bases has been already considered. By 
oxidation uric acid is transformed into urea or into substituted ureas, 
such as allantoin and alloxan, which latter in turn is closely related 
to parabanic acid or oxalyl-urea and barbituric acid or malonyl-urea. 



C 5 H 4 N 4 3 

Uric acid. 

C 5 H 4 N 4 3 

Uric acid. 



O -f H 2 



H 2 + 



C 4 H 2 NA 

Alloxan. 



C0<r NH 2 
Urea. 



C 4 H 6 N 4 3 + C0 2 . 
Allantoin. 



Pure uric acid forms a white crystalline powder which is almost 
insoluble in cold water (1 : 40,000), with difficulty soluble in boiling 
water (1 : 1800), and insoluble in alcohol and ether. In concentrated 
sulphuric acid it dissolves with ease, but is precipitated upon dilu- 
tion with water. In aqueous solutions of the alkaline carbonates 
and hydrates it dissolves, with the formation of neutral or acid salts, 
as represented by the equations : 

C 5 H 4 N 4 3 + Na 2 C0 3 = C 5 H 3 NaN 4 3 + NaHC0 3 . 
C 5 H 4 N 4 3 + 2Na 2 C0 3 = C 5 H 2 Na 2 N 4 3 + 2NaHC0 3 . 

In freshly voided urine uric acid is said to occur as a quadriurate, 
viz., as a compound in which one molecule of sodium is in combina- 
tion with two molecules of uric acid. The quadriurate, however, is 
readily decomposed with the formation of uric acid and acid urates 

Fig. 107. 




0®i 









Various forms of uric acid crystals. (Finlayson.) 



(biurates). Its solubility in the urine depends upon the amount of 
water present, the reaction^ and the presence of inorganic salts. 



CHEMISTRY OF THE URINE. 449 

When acid sodium phosphate preponderates, the biurate is precipi- 
tated, while free uric acid is thrown down when disodic phosphate 
only is present, and along with this still other acid compounds which 
are most likely of organic nature. Neutral urates cannot occur in 
the urine. The basic substances which may occur in the urine in 
combination with uric acid are sodium, potassium, ammonium, and 
possibly also calcium and magnesium. These salts may be decom- 
posed by the addition of a sufficiently large quantity of a stronger 
acid, such as hydrochloric acid, when uric acid is set free. The acid 
salts are soluble with great difficulty, and are hence precipitated 
whenever the urine is markedly acid or concentrated, and also when 
it is exposed to a low temperature. This holds good especially for 
the acid ammonium compound, and upon this fact Hopkins' quan- 
titative estimation of uric acid is based. 

Pure uric acid crystallizes in transparent, colorless, rhombic 
plates, while that which usually separates from the urine is of a 
reddish-brown color and may assume a great variety of forms (Fig. 
107). Of these, the so-called whetstone-form is the most character- 
istic (see Sediments). Colorless rhombic platelets may, however, 
also be seen. 

Of the compounds which uric acid forms with the heavy metals, 
the silver salt is especially important. When a solution of uric acid 
in ammonia is treated with an ammoniacal solution of silver nitrate 
(see below) the solution remains clear ; but if calcium chloride, 
sodium chloride, or magnesia mixture is then added, a precipitate 
forms, which contains the uric acid in combination with silver. 

Tests for Uric Acid. — 1. Murexid Test. — A few crystals are dis- 
solved by means of a few drops of concentrated nitric acid, with the 
application of heat, upon a porcelain plate, such as the cover of a 
crucible. The nitric acid is then carefully evaporated, when a yel- 
lowish-red spot will remain. Upon cooling, a drop of ammonia is 
placed upon this spot, when in the presence of uric acid a beautiful 
purplish-red color develops, owing to the formation of ammonium 
purpurate (murexid). If now a drop of sodium hydrate solution is 
added, the color changes to a reddish blue, which disappears upon 
heating ; the reaction thus differs from the somewhat similar xanthin 
reaction. 

2. Copper Test. — A few crystals are dissolved in sodium hydrate 
solution and treated with a few drops of Fehling's solution. Upon 
the application of heat white copper urate separates out, while red 
cuprous oxide appears if a relatively large amount of cupric sulphate 
is present — a point to be remembered in testing for sugar. The 
reduction of Fehling's solution is due to the formation of allantoin. 

3. When treated with sodium hypobromite solution uric acid gives 
up about 47 per cent, of its nitrogen. 

Quantitative Estimation of Uric Acid. — Hopkins' Method. — 
This method is now commonly used in the clinical laboratory, 
29 



450 THE UEINE. 

and is to be preferred to the more complicated procedures hitherto 
employed. It is much simpler and equally as accurate as the older 
methods of Ludwig-Salkowski and of Haycraft. Various modi- 
fications of the original method have been suggested. 

Principle. — The method is based upon the complete precipitation 
of uric acid by ammonium salts, and the possibility of accurately 
titrating the uric acid with potassium permanganate in the presence 
of sulphuric acid. 

Folin's Modification of Hopkins' Method. 1 — To precipitate the uric 
acid, and also to remove the small amount of mucoid substance 
which is found , in every urine, the following reagent is employed : 
500 grammes of ammonium sulphate and 5 grammes of uranium 
acetate are dissolved in 650 c.c. of water, to which solution 60 c.c. of 
a 10 per cent, solution of acetic acid are further added. The resulting 
solution measures about 1000 c.c. Seventy-five c.c. of the reagent are 
added to 300 c.c. of urine in a flask holding 500 c.c. After standing 
for five minutes the mixture is filtered through two folded niters, and 
thus freed from the mucoid body, which is carried down with the 
uranium phosphate in acid solution. The nitrate is divided into two 
portions of 125 c.c. each, which are placed in beakers and treated 
with 5 c.c. of concentrated ammonia. After stirring a little the solu- 
tions are set aside until the next day. The supernatant fluid is then 
carefully poured off through a filter (Schleicher and Schull, ISTo. 597) ; 
the precipitated ammonium urate is collected with the aid of a small 
amount of a 10 per cent, solution of ammonium sulphate and washed 
with the same reagent. Traces of chlorides do not interfere with 
the subsequent titration, and the process of filtration and washing 
can be completed in from twenty to thirty minutes. The ammonium 
urate is washed into a beaker, after opening the filter, using about 
100 c.c. of water. Fifteen c.c. of concentrated sulphuric acid are 
then added, and the solution is titrated at once with a one-twen- 
tieth normal solution of potassium permanganate. Toward the end 
of the titration Folin suggests to add the permanganate in portions 
of two drops at a time, until the first trace of a rose color is apparent 
throughout the entire fluid. Each cubic centimeter of the reagent 
corresponds to 0.00375 gramme of uric acid. A final correction of 
0.003 gramme for each 100 c.c. of urine employed is necessary, 
owing to the slight extent to which ammonium urate is soluble. 

Preparation of the One-twentieth Normal Solution of Potassium 
Permanganate. — As the molecular weight of potassium perman- 
ganate is 157.67, one would expect that a normal solution of the 
salt should contain this amount in grammes dissolved in 1000 c.c. 
of water. But the substance generally acts in the presence of free 
acids, upon deoxidizing substances, by losing 5 atoms of oxygen 
of the 8 atoms contained in 2 molecules, as is seen in the following 
equation : 

1 O. Folin u. A. Shaffer, Zeit. f. physiol. Chem., vol. xxxii. p. 552. 



CHEMISTRY OF THE URINE. 451 

2KMn0 4 + 5H 2 C 2 4 + 3H 2 S0 4 = K,S0 4 -f 2MnS0 4 + 10CO 2 + 8H 2 0. 

It follows that two-fifths of the molecular weight, or 63.068 
grammes, are the equivalent of 1 oxygen atom. But as oxygen 
is diatomic and the volumetric normal is calculated for monatomic 
values, this number must be divided by 2, and 31.534 grammes 
of potassium permanganate should therefore be present in 1 liter 
of normal solution. A one-tenth normal solution would hence 
contain 3.1534 grammes, and a one-twentieth normal solution 
1.576 grammes pro liter. This amount is weighed off and dis- 
solved in 950 c.c. of water, when the solution is brought to the 
proper degree of dilution (see page 392) by titration with a one- 
twentieth normal solution of oxalic acid. A one-twentieth normal 
solution of oxalic acid contains 3.142 grammes of the acid in 1000 
c.c. of water. One c.c. of the one-twentieth normal solution of 
potassium permanganate should correspond to 1 c.c. of the oxalic 
acid solution. The titration is best conducted by diluting 10 c.c. 
of the oxalic acid solution to 100 c.c. with distilled water and add- 
ing 15 c.c. of concentrated sulphuric acid, so as to bring the tempera- 
ture of the liquid to from 55° to 65° C. The potassium perman- 
ganate solution is then added drop by drop until the red color no 
longer disappears on stirring, but persists for at least thirty seconds. 

Titration with Sodium Hydrate Solution. — This method is not as 
accurate as the one just described, but suffices for ordinary purposes. 
The uric acid is precipitated with an ammonium salt, as above. 
After standing for two hours the ammonium urate is filtered off, 
washed with a 10 per cent, solution of ammonium sulphate, and 
brought into a beaker with the aid of a small amount of hot water. 
The salt is then decomposed by the addition of from 10 to 15 c.c. 
of a one-tenth normal solution of hydrochloric acid. The mixture 
is brought to • the boiling-point, and the hydrochloric acid not used 
in the decomposition of the ammonium urate retitrated with a one- 
tenth normal solution of sodium hydrate, using dimethyl -amido- 
azo-benzol as an indicator. The amount of hydrochloric acid found 
is deducted from the 10 or 15 c.c. added, and the result multiplied 
by 0.0168. The amount of uric acid contained in the original 
quantity of urine is thus ascertained, to which 0.003 gramme is 
added for each 100 c.c. of urine used, as above. 

Gravimetric Method. — The process is begun as described above. 
The ammonium urate is decomposed by the addition of from 2 to 3 
c.c. of a 25 per cent, solution of hydrochloric acid. This solution 
is evaporated until crystals of uric acid begin to separate out. These 
are collected on a dried and weighed filter, and washed successively 
with water, alcohol (90-95 per cent.), and absolute alcohol, and 
finally with ether. The mother-liquor and water used in washing 
are carefully measured, and 0.0004 gramme added to the final result 
for each 10 c.c. 



452 THE URINE. 

Haycraft's Method. 1 — This method is based upon the precipitation 
of uric acid with an ammoniacal silver solution and magnesia mixt- 
ure, 1 molecule of silver corresponding to 1 molecule of uric acid. 
As the amount of silver thus precipitated can be determined by titra- 
tion with a solution of potassium sulphocyanide, the corresponding 
amount of uric acid is readily found. 

Solutions required : 1. An ammoniacal silver solution. 2. An 
ammoniacal magnesia mixture. 3. A one-fiftieth normal solution 
of silver nitrate. 4. A one-fiftieth normal solution of potassium 
sulphocyanide. 

Preparation of these solutions : 

1. The ammoniacal silver solution is prepared by dissolving 26 
grammes of silver nitrate in distilled water, and adding enough 
ammonia to redissolve the brown precipitate of argentic oxide which 
is first formed ; distilled water is then added in sufficient amount to 
make the total quantity 950 c.c. This solution is brought to the 
proper strength by titrating a known amount of sodium chloride, as 
described elsewhere. Each cubic centimeter then contains 0.026 
gramme of silver nitrate, which is equivalent to 0.0169 gramme of 
silver. 

2. The ammoniacal magnesia mixture is prepared by dissolving 
100 grammes of crystallized magnesium chloride in a sufficient 
amount of water ; to this a cold saturated solution of ammonium 
chloride is added in excess, and sufficient strong ammonia to impart 
a decided odor. Should the mixture not be perfectly clear, more 
ammonium chloride solution is added. The solution is then diluted 
with water to 1 liter. 

3. The one-fiftieth normal solution of silver nitrate is prepared 
by dissolving 3.4 grammes of silver nitrate in 950 c.c. of distilled 
water, the degree of further dilution being determined as described 
elsewhere. 

4. To prepare the one-fiftieth normal solution of potassium sul- 
phocyanide, about 2 grammes of the salt are dissolved in 950 c.c. 
of water ; the solution is brought to the required strength, so that 1 
c.c. shall correspond to 1 c.c of the silver solution. 

For filtering the uric acid, a perforated platinum cone is placed in 
a small funnel and packed with a thin layer of glass-wool, upon 
which in turn a layer of finely scraped asbestos is placed. The 
asbestos is previously thoroughly washed with dilute hydrochloric 
acid and subsequently with distilled water until every trace of chlo- 
rine has disappeared. When properly prepared, the asbestos forms 
a mould of the cone. 

Method. — Five c.c. of the ammoniacal silver solution are mixed 
with 5 c.c. of the ammoniacal magnesia mixture. Ammonia is then 
added until the solution is clear, Avhen it is poured into 50 c.c. of 
urine. As soon as the precipitate has settled the supernatant liquid 

1 Haycraft, Zeit. f. analyt. Chem. ; vol. xxv. 



CHEMISTRY OF THE URINE. 453 

is passed through the prepared filter with the aid of a suction-pump. 
About 4 grammes of sodium bicarbonate in coarse pieces are now 
placed upon the filter and the precipitate added ; the sodium bicar- 
bonate serves the purpose of aiding filtration by loosening the pre- 
cipitate. This is now washed free from chlorine and silver by means 
of ammoniacal water, using the suction-pump until the precipitate 
appears broken in places, then without the pump, using this only at 
last to remove the last drops of liquid. (Test for silver with very 
dilute hydrochloric acid, and for chlorine with a solution of silver 
nitrate and nitric acid.) The precipitate is now dissolved on the 
filter by means of nitric acid of 20 to 30 per cent. The nitric 
acid must be free from nitrous acid. This is secured by allow- 
ing it to stand in contact with pure urea until any evolution of 
gas has ceased. The filter is washed with very dilute nitric acid 
and then with distilled water until this no longer shows an acid 
reaction. The solution thus obtained is titrated with the one-fiftieth 
normal solution of potassium sulphocyanide, using ammonio-ferric 
alum as an indicator. As each cubic centimeter of this solution 
indicates 0.0169 gramme of silver, and as 1 molecule of silver 
indicates 1 molecule of uric acid — i. e., 108 grammes of silver 168 
grammes of uric acid — 0.0169 gramme of silver, corresponding to 
1 c.c. of the potassium sulphocyanide solution, represents 0.2629 
gramme of uric acid. 

Ludwig-Salkowski Method. — Principle. — The method is based upon 
the formation of insoluble magnesium-silver urate when a solution 
of uric acid in sodium carbonate is treated with a solution of silver 
nitrate after the previous addition of an excess of ammonia. This 
is then decomposed, with the liberation of free uric acid. 

Method. 1 — Two hundred and fifty c.c. of urine are treated with 
50 c.c. of ammoniacal magnesia mixture (see above) to remove the 
phosphates. The magnesia mixture is employed for the reason that 
the compound of uric acid with magnesium and silver which is 
formed later on is not decomposed so easily as the sodium or the 
potassium compound, which would occur if the urine were pre- 
cipitated only with ammonia. The mixture is then immediately 
filtered, as otherwise a little magnesium urate would be precipitated. 
Two hundred and forty c.c. of the filtrate, corresponding to 200 c.c. 
of urine, are measured off as soon as possible, and treated with a 
few cubic centimeters of a 3 per cent, solution of silver nitrate. If 
the precipitated silver chloride formed in the beginning does not dis- 
appear on stirring, a little more ammonium hydrate is added. A 
flaky precipitate next separates out, and is allowed to settle. In 
order to test whether enough of the silver nitrate solution has been 
added, a few cubic centimeters of the supernatant fluid are acidified 
with nitric acid. If a distinct cloudiness, referable to silver 

1 E. Salkowski, Salkowski u. Leube, Die Lekr vom Harn. E. Ludwig, Wien. med. 
Jahrbiicher, 1884, p. 597. 



454 THE URINE. 

chloride, appears, enough has been added. Otherwise the few cubic 
centimeters that were employed for this test are rendered alkaline 
again with ammonia, poured back, and treated with more silver 
solution until the required amount has been reached. The liquid 
is then rapidly filtered through a folded filter of rather loose paper, 
a feather or rubber-tipped glass rod being used for the purpose of 
removing all the precipitate from the beaker. The precipitate is 
washed with ammoniacal water until a specimen of the washings 
is no longer rendered turbid by nitric acid, and only faintly so by 
the addition of a drop of silver solution. The filter with the pre- 
cipitate is next placed in a wide-mouthed flask, containing about 
200 c.c. of distilled water, and thoroughly agitated. Hydrogen 
sulphide is then passed through the mixture. It is now brought to 
the boiling-point and rendered distinctly acid by means of a few 
drops of hydrochloric acid, when the argentic sulphide and the 
paper are rapidly filtered off, as otherwise there will be an admixture 
of sulphur with the uric acid. The contents of the filter are washed 
a few times with hot water. Filtrate and washings are quickly 
evaporated to a few cubic centimeters, treated with a few drops of 
hydrochloric acid, and set aside in a cool place for twenty-four 
hours. Occasionally it happens that upon addition of the hydro- 
chloric acid a cloudiness appears, which is due to an admixture of 
sulphur. In such a case the dried uric acid must be washed with 
carbon disulphide. Otherwise the uric acid that has separated out 
is directly collected on a dried and weighed filter, and washed suc- 
cessively with water, 90 to 94 per cent, alcohol, and finally with 
absolute alcohol and ether. The water used in washing should be 
collected separately, and for each 20 c.c. used 0.0048 gramme added 
to the weight of the uric acid obtained. 

Precautions : 1. Rapidity in working is essential. 

2. Very concentrated urines must be diluted one-half before com- 
mencing the examination. 

3. If the specific gravity of the urine is low, it should be con- 
centrated to a specific gravity of about 1.020. 

4. If the urine shows a sediment of uric acid, this should be 
separately collected and weighed, and the weight obtained added to 
the final result. 

5. Any albumin that may be present must be previously removed. 

6. If sugar is present in the urine, about 500 to 1000 c.c. are 
treated with a solution of neutral lead acetate, filtered, and the 
filtrate precipitated with mercuric acetate. The precipitate thus 
formed, which consists essentially of mercuric urate, is filtered off 
after having stood for twelve to twenty-four hours, then washed, and 
later suspended in water. The mercury is removed by means of 
hydrogen sulphide, the mercuric sulphide filtered off, and the filtrate 
collected and set aside. The precipitate itself is thoroughly boiled 
with water and again filtered, the washings thus obtained being 



CHEMISTRY OF THE URINE. 455 

added to the filtrate set aside, as just described. The total amount 
of fluid is then evaporated to a small volume and acidified with 
hydrochloric acid, when the uric acid will separate out and may be 
treated as previously directed. 

The Xanthin-bases. 

The xanthin-bases which have been found in the urine are xanthin, 
hypoxanthin, heteroxanthin, paraxanthin, guanin, and adenin. Con- 
jointly they are also spoken of as the alloxur bases, or purin bases. 
Together with - uric acid they are termed alloxur or purin bodies. 
Their relation to uric acid and the nucleins has already been con- 
sidered (see page 442). Unlike uric acid, they also occur as such 
in animal as well as vegetable tissues. The amount which appears 
in the urine under normal conditions is very small, constituting 
about 10 per cent, of the uric acid. Larger quantities may be met 
with in various diseases, and, generally speaking, an increase in the 
amount of uric acid is associated with an increase of the xanthin- 
bases. This is, however, not invariably the case, and at times it 
may be observed that an increase of the uric acid is accompanied by 
a diminution of the xanthins, and vice versa. These varying rela- 
tions can, of course, be readily understood if we remember that uric 
acid is an oxidation-product of the xanthin-bases, and that their 
ultimate origin is the same. 

The literature which deals with the elimination of the xanthin- 
bases in various diseases has during the past few years assumed 
enormous proportions. This has largely been owing to the publica- 
tion by Kriiger and WulfF of a relatively simple method for their 
quantitative estimation. Unfortunately, however, this method has 
proved unreliable and the results obtained incorrect. Our knowl- 
edge of the relation of the xanthins to pathological processes is 
hence as defective at the present time as it was years ago. 

Individually the xanthin-bases are of little clinical interest. 
Xanthin has once been found in a urinary sediment, and has in 
several instances been encountered as the principal constituent of 
vesical calculi. Its normal quantity is said to vary between 0.02 
and 0.03 gramme. Larger quantities are found after a meal rich 
in nucleins, in leukaemia, nephritis, pneumonia, etc. 

Paraxanthin and heteroxanthin are present only in traces, as is 
apparent from the fact that Kriiger and Salomon were able to obtain 
but 7.5 grammes of heteroxanthin from 10,000 liters of urine. 
Both apparently are distinctly toxic. 

Xanthin sediments may be recognized by means of the following 
test : a small amount of the material is treated with a few drops of 
concentrated nitric acid on a porcelain plate, and evaporated to dry- 
ness. In the presence of xanthin a yellow residue is obtained, which 
turns red upon the addition of a few drops of sodium hydrate solu- 



456 THE URINE. 

tion and the application of heat. The reaction is common to all the 
xanthins. 

Quantitative Estimation. — Salkowski's Method. 1 — Six hundred 
c.c. of urine are precipitated with 200 c.c. of magnesia mixture 
(see page 401), when a 3 per cent, ammoniacal solution of 
silver nitrate is added to from 700 to 750 c.c. of the nitrate. 
The proportion should be 6 c.c. for each 100 c.c. of urine. 
The silver nitrate solution should be added as described on 
page 453. After standing for one hour the mixture is filtered, and 
the precipitate washed with water until all the free silver has been 
removed. The filter is then perforated, the precipitate washed into 
a flask with from 600 to 800 c.c. of water, acidified with hydro- 
chloric acid, and decomposed with hydrogen sulphide. The excess 
of hydrogen sulphide is removed by heating on a water-bath, when 
the silver suphide is filtered off and the filtrate evaporated to dryness. 
The residue is treated with from 25 to 30 c.c. of dilute sulphuric 
acid (1 : 100). This solution is brought to the boiling-point and is 
allowed to stand over night. The uric acid which has then sepa- 
rated out is filtered off, washed with a small amount of dilute sul- 
phuric acid (not more than 50 c.c), then with alcohol and ether, and 
weighed. To the resulting weight 0.0005 gramme is added for 
each 10 c.c. of the acid filtrate, to allow for the trace of uric acid 
which is thus lost. 

After having filtered off the uric acid the filtrate is again treated 
with ammonia and silver solution, and the xanthin-bases thus pre- 
cipitated. The precipitate is collected on a small filter, washed with 
water, dried, and incinerated. The ash is dissolved in nitric acid, 
and the silver estimated by titration with a solution of potassium 
sulphocyanide, using ammonio-ferric alum as an indicator (see page 
393). The solution of potassium sulphocyanide employed in the 
estimation of the chlorides may be used, and is of such strength 
that 1 c.c. corresponds to 0.00734 gramme of silver. As 1 atom 
of silver in a mixture of the silver compounds of guanin, xanthin, 
hypoxanthin, etc., represents 0.277 gramme of nitrogen, or 0.7381 
gramme of the alloxur bases, it is apparent that 1 c.c. of the potas- 
sium sulphocyanide solution will represent 0.002 gramme of nitro- 
gen and 0.00542 gramme of alloxur bases. In every case an accu- 
rate record must, of course, be kept of the amount of urine and 
filtrate used. 

The amount of alloxur bases found by Salkowski in the normal urine 
of twenty-four hours varied between 0.0286 and 0.0561 gramme. 

Literature.— M. Kriiger u. G. Salomon, "Die Alloxurbasen d. Hams," Zeit. f. 
pbysiol. Cbem., vol. xxiv. p. 364, and vol. xxvi. p. 343; Deutsch. med. Woch., 1899, p. 
97. Bondsynski u. Gottlieb, " Ueber Xanthinkorper im Harn des Leukamiker," Arcb. 
f. exper. Patb. u. Pbarmakol., 1895, vol. xxxvi. p. 132. F. Guinprecht, " Alloxurkorper 
u. Leukocyten," Centralbl. f. allg. Patb. u. patb. Anat., 1896, vol. vii. p. 820. 

1 E. Salkowski, Pfliiger's Arcbiv, vol. lxix. p. 268. 



CHEMISTRY OF THE URINE. 457 

Hippuric Acid. 

Hippuric acid is a constant constituent of normal urine, 0.1 to 1 
gramme being excreted in the twenty-four hours. That it is derived, 
to some extent at least, from albuminous material is proved by the 
fact that its elimination is not suspended during starvation nor during 
the administration of a purely albuminous diet. The manner in 
which hippuric acid is formed in the body-economy, however, has 
not been definitely ascertained. In vitro it may be obtained from 
glycocoll and benzoic acid, according to the equation 



C 6 H 5 CH 2 NH 3 

! + 1 

COOH COOH 


CH 2 NH — C 6 H 5 CO 
= 1 +H 3 0. 
COOH 


Benzoic acid. Glycocoll. 


Hippuric acid. 



It has been shown that phenyl-propionic acid, which differs from 
benzoic acid by the group C 2 H 4 , and which latter may be regarded 
as phenyl-formic acid, is produced during the process of intestinal 
putrefaction. The relation between the two bodies is seen from the 
formulae : 

H C 6 H 5 CH S CH 2 .C 6 H 5 

| m-> | | | 

COOH COOH CH 2 i»— > CH 2 

Formic Phenyl-formic 

acid. acid. COOH COOH 

Propionic Phenyl-propionic 
acid. acid. 

Phenyl-propionic acid is then absorbed into the blood and there, 
according to our present ideas, transformed into phenyl-formic acid 
or benzoic acid. When the latter comes in contact with glycocoll, 
w T hich is probably also produced during the process of intestinal 
putrefaction, an interaction between the two substances occurs in 
the body, hippuric acid resulting, as shown in the above equation. 
This view is supported by the fact that phenyl-propionic acid, just 
as benzoic acid, when introduced into the circulation of certain ani- 
mals, reappears in the urine as hippuric acid. The final proof of 
the possible synthesis of hippuric acid from glycocoll and benzoic 
acid in the body has been furnished by Bunge and Schmiedeberg, 1 
who obtained this substance, when arterialized blood containing 
glycocoll and sodium benzoate was allowed to pass through isolated 
kidneys of dogs. 

Not all the hippuric acid eliminated, however, is referable to albu- 
minous decomposition, but a considerable portion is derived from 
benzoic acid or its derivatives, which occur in many fruits, and 
are transformed into hippuric acid in the body. Among those 
which are particularly rich in these substances may be mentioned 

1 Schmiedeberg u. Bunge, Arch. f. exper. Path. u. Pharmakol., vol. vi. 



458 THE URINE. 

the red bilberry, prunes, coffee-beans, reinesclaudes, etc., and in all 
cases in which an increased elimination of hippuric acid is observed 
the possibility of this source must always be taken into account. 

As to the seat of this synthesis there appears to be some uncer- 
tainty, as it is apparently not the same in all animals. In the dog 
and the frog the kidneys, according to the researches df Bunge and 
Schmiedeberg, must be regarded as the principal and possibly the 
only organs in which this process occurs. As Salomon, however, 
has demonstrated the presence of hippuric acid in the muscles, liver, 
and blood of nephrectomized rabbits, still other organs must, in the 
herbivora at least, be concerned in its production. 

Very little is known of the pathological variations in the excre- 
tion of hippuric acid ; this is principally owing to the fact that until 
recently suitable methods for its quantitative estimation were not 
available. It is an interesting fact that, in accordance with Bunge's 
experiments in dogs, the formation of hippuric acid appears to be 
suspended in cases of acute as well as chronic parenchymatous 
nephritis, for the benzoic acid which is then ingested reappears 
in the urine unchanged. In amyloid degeneration a marked dimi- 
nution in its amount has likewise been demonstrated. Large quan- 
tities of hippuric acid, on the other hand, have been noted in acute 
febrile diseases, hepatic diseases, diabetes mellitus, chorea, etc. The 
data, however, are insufficient to warrant any definite conclusions at 
the present time. 1 

Properties of Hippuric Acid. — Chemically, hippuric acid must 
be regarded as benzoyl-amido-acetic acid, C 9 H 9 N0 3 — (C 6 H 5 .CONH. 
CH 2 .COOH). It crystallizes in long rhombic prisms when allowed 
to separate from its solutions gradually, while it forms long needles 
if crystallization takes place rapidly and the amount is small (Fig. 
95). In cold water and ether it is soluble with difficulty, while it 
dissolves readily in hot water, in alcohol, and in aqueous solutions 
of the hydrates and carbonates of the alkalies, with which it forms 
salts, and from which the acid may again be separated and caused 
to crystallize out by adding a stronger acid. 

When hippuric acid or one of its salts is evaporated to dryness 
with concentrated nitric acid and the residue is heated, the odor of 
bitter almonds is noticed ; this is due to the formation of nitro- 
benzol. 

When boiled with hydrochloric acid or dilute sulphuric acid 
hippuric acid is decomposed into glycocoll and benzoic acid. A 
similar decomposition is effected during the process of putrefaction, 
and hence no hippuric acid is found in decomposing urine, benzoic 
acid taking its place. The latter is always found in the urine 
together with hippuric acid, but has no clinical significance. In 

1 Th. Weyl u. B. von Anerep, " Ueber die Ausscheidung der Hippursaure und Ben- 
zoesaure wahrend des Fiebers," Zeit. f. pbysiol. Chem., 1880, vol. iv. p. 169. 



CHEMISTRY OF THE URINE. 



459 



larger amounts it has recently been encountered in a case of diabetes. 
It crystallizes in needles or lustrous laminae, presenting ragged 
edges, which resemble plates of cholesterin. It is soluble with 
difficulty in cold water, but easily soluble in ether, alcohol, and solu- 
tions of the alkaline carbonates and hydrates, forming salts with the 
latter, 

Hippuric acid in the urine occurs in combination with sodium, 
potassium, calcium, and magnesium. 

Quantitative Estimation of Hippuric Acid. — The following 
method, which may be employed for the quantitative estimation of 
hippuric acid, although tedious, must also be employed when it is 
desired to test for its presence. 

Principle. — Hippuric acid readily dissolves in solutions of the 
alkaline hydrates and carbonates, forming salts. These are decom- 
posed by means of a stronger acid, when the hippuric acid which 
separates out is collected and weighed. 



Fig. 108. 




Hippuric acid crystals. 

Method. — Five hundred to 1000 c.c. of fresh urine are evap- 
orated to a syrupy consistence on a water-bath, care being taken to 
keep the urine neutral by the addition of sodium carbonate. The 
residue is extracted with cold alcohol (90 to 95 per cent.), using 
about half of the quantity as that of the urine employed. The 
mixture is then set aside for twenty-four hours. The alcoholic 
nitrate, which contains the salts of hippuric acid, is freed from 
alcohol by distillation. The remaining solution is strongly acidified 
with acetic acid and extracted with at least five times its volume of 
alcoholic ether (1 part of alcohol to 9 parts of ether). From the 
combined extracts the ether is distilled off and the remaining solu- 
tion evaporated on a water-bath. The resinous residue is boiled 
with water, set aside to cool, and filtered through a well-moistened 



460 THE URINE. 

filter. The hippuric acid, which is easily soluble in boiling water, 
is thus separated from other constituents which are soluble in alco- 
hol and ether. The filtrate is rendered alkaline with a little milk 
of lime, any excess of calcium being removed by passing carbon 
dioxide through the mixture. This is then brought to the boiling- 
point and filtered. Any impurities which may be present are re- 
moved by shaking with ether. The calcium salts remaining in solu- 
tion are decomposed by means of an acid, when the solution is again 
extracted with ether. The remaining solution is evaporated to a few 
cubic centimeters, when the hippuric acid will separate out on stand- 
ing. The crystals are dried on plates of plaster of Paris, shaken 
with benzol or petroleum-ether to remove any benzoic acid, and 
finally weighed. These crystals may be shown to be hippuric acid 
by their microscopical appearance, their solubility in alcohol, and 
their behavior when evaporated with concentrated nitric acid, as 
indicated above. 

Hofmeister's Method. — Two hundred to 300 c.c. of urine are evap- 
orated in a glass dish to one-third of the original volume, and 
treated with 4 grammes of disodium phosphate, to transform the 
acid into its sodium salt. The mixture is evaporated to a syrupy 
consistence, the residue treated with burnt gypsum, dried thoroughly, 
and pulverized together with the dish. The powder is extracted in 
a Soxhlet apparatus with freshly rectified petroleum-ether (boiling- 
point 60° to 80° C.) for forty-six hours, and then for six to ten 
hours with pure ether (free from water and alcohol). After dis- 
tilling off the ether the residue is dissolved in boiling water and 
decolorized with animal charcoal, the latter being subsequently 
thoroughly washed with boiling water ; the solution and washings 
are evaporated to about 1 or 2 c.c. at a temperature of from 50° to 
60° C, and set aside to crystallize. The crystals of hippuric acid 
are finally washed with a few drops of water and ether, and weighed. 

Kreatin and Kreatinin. 

Kreatin, which is constantly present in muscle-tissue, is in all 
probability the immediate and constant antecedent of kreatinin, so 
that two sources of this body must be recognized, viz., the muscle- 
tissue of the body and the muscle-tissue ingested as food. Beyond 
this, however, practically nothing is known, and as the artificial pro- 
duction of kreatinin from albuminous material has never been 
accomplished, it is hardly warrantable to venture an hypothesis as to 
its mode of formation in the body. 

Kreatinin is a constant constituent of the urine, about 1 gramme 
being excreted daily by a healthy adult. Pathologically, variations 
in this amount have been observed, but the data obtained possess 
little value. Before drawing conclusions from observations made in 



CHEMISTRY OF THE URINE. 461 

the clinical laboratory it is necessary to take into account the quan- 
tity of meat ingested, as a meat-diet will greatly increase the amount 
of kreatinin. If then in patients affected with acute febrile dis- 
eases, such as pneumonia, typhoid fever, etc., a large increase is 
observed, the patient being at the same time upon a milk-diet, an 
increased destruction of muscle-tissue may be inferred, as a milk- 
diet in itself, cceteris paribus, causes a diminished elimination. A 
decrease would logically be expected to occur during convalescence 
from such diseases. In the various forms of anaemia, marasmus, 
chlorosis, phthisis, etc., a diminished amount is observed. 1 

The transformation of kreatin into kreatinin has been supposed to 
take place in the kidneys, a view which accords with the greatly 
diminished excretion of kreatinin in advanced cases of chronic 
parenchymatous nephritis. In progressive muscular atrophy, in 
pseudohypertrophic paralysis, and in progressive ossifying myositis 
a diminution has also been noted. 

Properties of Kreatin and Kreatinin. — Chemically, kreatin may 
be regarded as a methyl derivative of glucocyamin, which latter is 
guanidin in which 1 NH 2 group has been replaced by glycocoll. 
Kreatinin, on the other hand, is the methyl derivative of glucocy- 
amidin, which differs from glucocyamin only in the absence of 1 
molecule of water, so that kreatinin is kreatin minus 1 molecule of 
water, both being derivatives of guanidin. The relation between 
the various bodies is shown below : 

/NH 2 

C=NH 

\NH 2 

Guanidin. 

/NH 2 /NH 2 

C=NH C==NH 

\NH. CH 2 .COOH \N(CH 3 ) . CH 2 .COOH 

Glucocyamin. Kreatin. 

/NH /NH 

C=NH C=N 

\NH.CH 2 .CO \N ( CH 3 ) .CH 2 .CO 

Glucocyamidin (glucocyamin minus water). Kreatinin (kreatin minus water). 

Kreatinin crystallizes without water of crystallization in colorless, 
glistening prisms. At times, when the crystals are not well devel- 
oped, it also appears in the form of whetstones. It is readily soluble 
in hot and also quite soluble in cold water and hot alcohol ; in cold 
alcohol and ether it dissolves with difficulty. It forms salts with 
acids, and double salts with some of the salts of the heavy metals. 
Among these may be mentioned kreatinin hydrochloride, C 4 H 7 N 3 0. 
HC1, which is easily soluble in water and crystallizes in the form of 
transparent prisms or rhombic plates. Most important is the com- 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co., 1901. Senator, Virchow's 
Archiv, 1876, vol. Ixvii. p. 422. Neubauer u. Vogel, Harnanalyse, Pt. ii. 



462 



CHEMISTRY OF THE URINE. 



pound of kreatinin with zinc chloride, (C 4 H 7 N s O) 2 .ZnCl 2 (Fig. 109). 
This is produced when a watery or alcoholic solution of kreatinin is 
treated with zinc chloride. The crystalline form of this compound 
depends greatly upon the purity of the kreatinin solution. When 
obtained from alcoholic extracts of the urine it occurs in the form 
of varicose conglomerations which often adhere firmly to the walls 
of the vessel. If the solution of kreatinin is perfectly pure, how- 
ever, it is seen in the form of fine needles grouped in rosettes or 
sheaves. Kreatinin-zinc chloride is soluble with much difficulty in 
water and insoluble in alcohol. The compound is especially impor- 
tant, as upon its formation and properties the quantitative estimation 
of kreatinin in the urine is based. Silver nitrate and mercuric chlo- 
ride cause a precipitation of kreatinin, and may, therefore, also be 
employed for the purpose of obtaining the substance from the urine. 

Fig. 109. 




Crystals of kreatinin-zinc chloride. (Salkowski.) 

Test for Kreatinin in the Urine. — A few cubic centimeters of 
urine are treated with a few drops of a very dilute solution of sodium 
nitroprusside and then drop hy drop with a dilute solution of sodium 
hydrate. In the presence of kreatinin the urine assumes a ruby-red 
color, which is particularly well seen in the lower portion of the 
tube. This color disappears after a few minutes, and is replaced by 
an intense yellow, which on warming with glacial acetic acid in pure 
solutions turns to green, then to blue, and on standing a deposit of 
Prussian blue is obtained (WeyVs test)? The presence of albumin 
or sugar does not interfere with the reaction. 

Quantitative Estimation of Kreatinin in the Urine. 2 — Prin- 
ciple. — When an alcoholic extract of urine is treated with an alco- 

1 Th. Wevl, Ber. d. deutsch. chem. Gesellsch., 1878, vol. xi. p. 217 ; and Jaffe, Zeit. 
f. physiol. Chem., 1886, vol. x. p. 399. 

2 Leube u. Salkowski, Die Lehre vom Harn, Hirschwald, Berlin, 1882, p. 111. 



CHEMISTRY OF THE URINE. 463 

holic solution of zinc chloride kreatinin-zinc chloride separates out. 
This, as has been mentioned, is almost insoluble in alcohol. Know- 
ing the molecular weight of kreatinin and kreatinin-zinc chloride, 
the calculation of the amount of kreatinin becomes a simple matter. 
The molecular weight of kreatinin is 113, that of kreatinin-zinc 
chloride 362. In 362 parts by weight of the latter there are, hence, 
226 parts by weight of kreatinin, so that the amount of the kreatinin 
may be calculated from the weight of the kreatinin-zinc chloride 
according to the following equation : 362 : 226 : : y : x ; and x — - 
0.6243 y, in which y indicates the weight of the kreatinin-zinc 
chloride found, and x the corresponding amount of kreatinin. The 
phosphates must, of course, first be eliminated, as insoluble zinc 
phosphate would otherwise be precipitated. 

Method. — In 200 c.c. of urine the phosphates are first removed 
by alkalinizing with milk of lime, and then adding calcium chloride 
so long as a precipitate forms. If the volume is now less than 
300 c.c, water is added to that amount. The mixture is filtered 
after having been allowed to stand for from one-quarter to one- 
half hour, and washed with a little water. Filtrate and washings 
are slightly acidified with dilute hydrochloric acid, so as to prevent 
the transformation of kreatinin into kreatin, and evaporated on 
a water-bath to a syrupy consistence, and then thoroughly mixed 
with 20 to 30 c.c. of absolute alcohol. The mixture is poured into 
a stoppered flask provided with a 100 c.c. mark, and after thor- 
oughly rinsing out the evapora ting-dish with absolute alcohol the 
washings are also placed in the bottle, and absolute alcohol is added 
to the mark. The bottle is thoroughly shaken and set aside in a cool 
place for twenty-four hours, the mixture being agitated from time to 
time. It is now filtered and rendered slightly alkaline with a drop 
or two of a sodium carbonate solution, as kreatinin hydrochloride is 
not precipitated by zinc chloride. The reaction, however, should 
be only faintly alkaline, as otherwise zinc oxide will be precipitated. 
The mixture is now slightly acidified with acetic acid and treated 
with 0.5 c.c. of an alcoholic solution of zinc chloride, prepared by 
dissolving the salt in 80 per cent, alcohol and diluting with 95 
per cent, alcohol to a specific gravity of 1.2. The mixture is well 
stirred and set aside in a cool place for two or three days. The 
crystals, which are usually deposited on the sides of the vessel in 
the form of wart-like masses, are then collected upon a dried and 
weighed filter, always using portions of the filtrate to bring the 
crystals completely upon the filter. These are washed with a small 
amount of 90 per cent, alcohol, until the washings are without color 
and give only a slight opalescence when treated with a drop of silver 
nitrate solution. The crystals are finally dried at a temperature 
of 100° C, and weighed. By multiplying the weight thus found by 
0.6243 the amount of kreatinin is obtained. 



464 THE URINE. 

Precautions : 1. Albumin and sugar, if present, must first be 
removed. In diabetic urines it is best, after having removed the 
sugar by fermentation, to take one-fifth of the total quantity elimi- 
nated in twenty -four hours, and to evaporate this to about 300 c.c. 
before removing the phosphates. 

2. The weighed material should be examined microscopically, 
to see whether notable quantities of sodium chloride are present. 
Should this be the case, it is necessary to determine the amount 
of zinc present, and to estimate the kreatinin from this. To this 
end, the kreatinin-zinc chloride is evaporated to dryness after the 
addition of a little nitric acid. The residue is incinerated, extracted 
with water, washed, dried, fused, and finally weighed. 

As 100 parts of kreatinin-zinc chloride correspond to 22.4 parts 
by weight of zinc oxide, the corresponding amount of the compound 
is found according to the following equation : 22.4: 100 : : y : x ; 
and x = 4.4642 y, in which y represents the amount of zinc oxide 
found, and x the corresponding amount of kreatinin-zinc chloride. 
By multiplying the number thus ascertained by 0.6243 the amount 
of kreatinin is found. 

3. Instead of doing this, the precipitate in the alcoholic solution 
may be examined microscopically before filtering. If sodium chlo- 
ride crystals are found, providing that the kreatinin-zinc chloride 
adheres to the sides of the vessel, the sodium chloride may be dis- 
solved in a little water and poured off. 

4. If the crystals of kreatinin-zinc chloride adhere very firmly 
to the sides of the vessel, so that their removal would be incomplete, 
it is perhaps best to dissolve them in a little hot water, to evaporate 
to dryness, and to weigh the kreatinin compound directly. 

5. If the urine shows an alkaline reaction, it is best to acidify 
with sulphuric acid, and to boil for half an hour before removing the 
phosphates, so as to transform any kreatin that may be present into 
kreatinin, when the examination is continued as described. 

Folin's Method. 1 — This method is based on Jaffe's reaction of 
kreatinin with alkaline picric acid solution. The red colored solu- 
tion produced in this reaction has in proper concentration and when 
viewed by transmitted light exactly the same shade as a potassium 
bichromate solution. Half-normal potassium bichromate solution 
(containing 24.55 grammes per liter) is therefore used as a standard 
for comparison. 

A high-grade colorimeter, by means of which the depths both of 
the unknown solution and of the bichromate can be adjusted to tenths 
of millimeters, is necessary for the comparison. 2 

1 The above description of the as yet unpublished method I owe to the courtesy of 
O. Folin. 

2 The French instrument of Duboscq, which can be obtained through Eimer & 
Amend, is admirably suited for the purpose. 



CHEMISTRY OF THE URINE. 465 

The following solutions are also necessary : The half-normal po- 
tassium bichromate solution, 10 per cent, sodic hydrate, and a satu- 
rated (1.2 per cent.) picric acid solution. 

If to 10 mgrms. of chemically pure kreatinin dissolved in 10 c.c. 
of water in a 500 c.c. volumetric flask are added 15 c.c. of picric 
acid solution and 5 c.c. of sodic hydrate, the maximum color is ob- 
tained at the end of five minutes. If at the end of this time the 
solution be diluted to the 500 c.c. mark and at once compared with 
the standard bichromate solution, it will be found that 8.1 mm. of 
the kreatinin-picrate solution have in the colorimeter exactly the 
same shade and depth of color as 8 mm. of the bichromate 
solution. 

The actual determination in urine is carried out in exactly the 
same way, substituting 10 c.c. of urine for the kreatinin solution. The 
more kreatinin that is present in the 10 c.c. of urine the deeper will, 
of course, be the color of the solution obtained. Supposing the 
colorimetric observation shows that 7.1 mm. of the urine picrate 
solution are equal in color to 8 mm. of the standard. The 10 c.c. 

of urine would then contain 10 X -— = 11.4 mgrms. of kreatinin. 

The following precautions are to be observed in the determination : 

1. Make first a preliminary colorimetric observation, using half- 
normal potassium bichromate solution in both cylinders of the col- 
orimeter, adjusting first one to the 8 mm. mark. The average of 
three or four readings of the other cylinder should also be 8 mm., 
and after the first observation no two should differ by more than 0.2 
mm. This preliminary observation takes only two or three minutes, 
and is exceedingly useful in making the eye sure of the correct point 
to be ascertained. 

2. Exactly 8 mm. of the half-normal potassium bichromate solu- 
tion must be used as the standard for comparison. 16 or 24 mm., 
for example, cannot be substituted on the basis of the calculation 
given above because the kreatinin picrate solution absorbs light at 
an entirely different rate from that of the bichromate solution. 

3. For the reason given in the preceding paragraph it is necessary 
to make each determination with a quantity of urine containing not 
less than 5 nor more than 15 mgrms. of kreatinin. Within these 
limits the determination as described is correct within 0.2 mgrm. 

4. Sugar and albumin do not interfere with the determination. 
Acetone, diacetic acid, and hydrogen sulphide do interfere. Where 
these are present the urine should be measured into a porcelain evap- 
orating-dish and heated on a water-bath with 10 c.c. of 1 per cent, 
hydrochloric acid for about half an hour. When the dish is again 
cooled, the reagents are added directly into the dish, and finally 
rinsed into the volumetric flask after five minutes. 

5. The color due to the urine is ordinarily of no appreciable con- 

30 



466 THE URINE. 

sequence because of the great dilution. Urines containing bile-pig- 
ments can, however, first be cleared by the addition of egg-albumin 
and then removing this by coagulation (heat). 

The whole operation can be finished in less than fifteen minutes ; 
indeed, it should be finished at once, as the colored product obtained 
by the interaction of kreatinin and picric acid is not very stable. 

Oxalic Acid. 

The origin of oxalic acid in normal urine is twofold. The greater 
portion is supposedly derived from the ingested food, but there 
is evidence to show that a certain amount is also formed during 
the metabolism of the body-tissues, as the elimination of oxalic acid 
does not cease during starvation. The carbohydrates and fats 
probably do not play a part in this connection ; and, according 
to Salkowski, the albumins also do not enter into consideration 
per se. He rather inclines to the view that the nucleins represent 
the antecedent of the oxalic acid, and as a matter of fact uric acid, 
which, as we have seen, is itself derived from the nucleinic bases, 
can be readily oxidized to oxalic acid, with the intermediary forma- 
tion of parabamic acid and oxaluric acid. The latter has been 
repeatedly demonstrated in the urine, and it is conceivable that the 
same process may occur in the animal body. But even suppos- 
ing that the oxaluric acid which is obtained from the urine is formed 
artificially during the lengthy process of analysis, and that the sub- 
stance did not exist preformed, there is no reason for the assump- 
tion that uric acid may not be the normal antecedent of the oxalic 
acid. For Salkowski has demonstrated conclusively that on oxida- 
tion with ferric chloride in aqueous solution uric acid yields oxalic 
acid and urea directly. These various changes may be expressed 
by the equations : 

NH.CO y NH 2 

(i) WA + h 2 o + 20 = co^ nh | Q + co/ nh2 + CO, 

Uric acid. Parabanic acid. Urea. 



.NH.CO CO.NH.CONH 2 

(2) C0< | +H 2 =| 

\NH.C0 COOH 

Parabanic acid. Oxaluric acid. 

CO.NH.CONH 2 CO.OH .NH 2 

(3) i +h 2 o =i +co<; 

V } COOH CO.OH X NH 2 

Oxaluric acid. Oxalic acid. Urea. 

CO.OH ,NH 2 

(4) WA +3H 2 + 20= J ^ W + 2C0 C TT + C0 * 

CO.OH X JNJ1 2 

Uric acid. Oxalic acid. Urea. 



CHEMISTRY OF THE URINE. 467 

The matter, however, is not quite so simple as it appears, and an 
increased elimination of oxalic acid by no means always occurs 
when the output of uric acid is increased. After the ingestion of 
fairly large amounts of thymus, for example, the usual increase of 
uric acid is not accompanied by a corresponding increase in the 
amount of oxalic acid, and in those cases in which it does occur 
we are as yet unable to exclude the large amount of connective 
tissue as the source of the oxalic acid. Connective tissue and 
gelatin have, as a matter of fact, been shown to increase the amount 
of oxalic acid when given in large amounts. With pure nuclein 
no effect has been observed, and it can be shown that in those 
experiments in which this was used by mouth an absorption from 
the intestinal tract had manifestly not occurred (Mohr and Salo- 
mon). 1 

Under pathological conditions oxalic acid may also be formed 
in the digestive tract from the ingested carbohydrates, as a re- 
sult of a peculiar fermentative process. This has been well shown 
by Helen Baldwin in Herter's laboratory. In some of these 
cases no free hydrochloric acid could be demonstrated in the gastric 
contents, and it was observed that inoculation of a digestive mixt- 
ure, which was originally free from oxalic acid, resulted in its ap- 
pearance if a few drops of such stomach contents were added. In 
dogs prolonged feeding with excessive quantities of glucose together 
with meat was seen to lead eventually to a state of oxaluria, which 
was associated with a mucous gastritis and the absence of free hydro- 
chloric acid. Oxalic acid could then also be demonstrated in the 
stomach contents. 

Very curiously the ingestion of quite small and non-toxic amounts 
of oxalic acid is followed by a fairly intense indicanuria. It does 
not seem likely to me, however, that as Harnack and v. d. Leyen 
suggest, the indicanuria is here referable to a toxic action upon the 
tissue-albumins, and I am personally inclined to explain the phe- 
nomenon upon the basis of increased intestinal putrefaction (see 
Indicanuria). 

The amount of oxalic acid which is normally eliminated in the 
twenty-four hours fluctuates with the amount ingested, and varies 
from a few milligrammes to 2 or 3 centigrammes, being usually less 
than 10 milligrammes (Baldwin). It is influenced by the character 
of the diet. The ingestion of oxalates by the mouth is followed 
only by their partial elimination in urine and feces, so that we 
may conclude that to a certain extent oxalic acid is decomposed 
during its passage through the animal body ; possibly this may occur 
in the intestinal canal as the result of bacterial action. 

Foods rich in oxalic acid are spinach, tomatoes, carrots, celery, 

1 L. Mohr and H. Salomon, Deutsch. Arch. f. klin. Med., 1901, vol. lxx. p. 486. 
Lommel, Ibid., vol. lxiii. p. 599. 



468 THE URINE. 

string-beans, rhubarb, potato, dried figs, plums, strawberries, 
cocoa, tea, coffee, and pepper. Foods which contain little or no 
oxalic acid, on the other hand, are meat, milk, eggs, butter, corn- 
meal, rice, peas, asparagus, cucumbers, mushrooms, onions, lettuce, 
cauliflower, pears, peaches, grapes, melons, and wheat, rye, and oat 
flour. 

Before drawing conclusions as to the existence of abnormal 
oxaluria it is hence imperative to eliminate the possibility of an 
increased ingestion, by placing the patient upon a diet which con- 
tains little or no oxalic acid. 

An increased elimination is notably observed in association with 
various dyspeptic and nervous manifestations, and constitutes the 
condition commonly spoken of as the oxalic acid diathesis, or as 
idiopathic oxaluria. Its existence as a definite pathological picture 
is, however, denied by most modern clinicians. Nevertheless it 
must be admitted that there is a certain type of neurasthenia in 
which, generally in association with hyperchlorhydria, an increased 
elimination of oxalic acid takes place, and in which a copious de- 
posit of calcium oxalate crystals is frequently observed. From the 
mere fact of the occurrence of such deposits, of course, no inference 
is as rule to be drawn regarding the actual elimination, but its fre- 
quent occurrence is in itself of importance, as in such cases a 
similar separation from the urine may already occur within the urinary 
passages, and not uncommonly in the pelvis of the kidneys. Not 
infrequently oxaluria of this type is associated with an increased 
elimination of uric acid and a mild grade of albuminuria, as has 
been shown by Senator, v. Noorden, DaCosta, myself, and others. 
Whether or not the oxaluria in these cases can be explained upon 
the basis of abnormal fermentations in the gastro-intestinal tract, 
as is suggested by the observations of Baldwin, remains to be seen. 
In some this may be the case, but in others I am inclined to asso- 
ciate the oxaluria with the coexistent lithuria, and rather imagine 
that both conditions may be referable to impairment of the normal 
oxidation-processes in the liver. 

That this explanation holds good also of the apparent vicarious 
oxaluria which is at times observed in diabetes, appears quite likely. 

Fiirbringer has reported a case of diabetes in which the elimi- 
nation of oxalic acid was described as " enormous," and in which 
oxalic acid could also be demonstrated in the sputum (oxaloptysis). 
Rausch has recorded a case of mild diabetes, associated with 
hepatic cirrhosis, in which 1.2 grammes were excreted in twenty- 
four hours. In most cases of diabetes, on the other hand, an in- 
creased oxaluria cannot be demonstrated. 

In cases of obesity Kisch found no abnormal degree of oxaluria. 

In association with jaundice increased oxaluria has been re- 
peatedly observed, and is probably referable to biliary stasis and 



CHEMISTRY OF THE URINE. 469 

consequent cholsemia, as Salkowski has demonstrated that the bile 
contains oxalic acid. In pneumonia and leukaemia, in both of which 
we find as a rule a greatly increased elimination of uric acid, the 
oxalic acid is not always increased, and sometimes indeed quite low 
in comparison to the amount of uric acid. 

Properties of Oxalic Acid. — Oxalic acid occurs in the urine as 
calcium oxalate, CaC 2 4 , and is held in solution by the diacid sodium 
phosphate. It can, hence, be precipitated by diminishing the acidity 
of the urine by the addition of a little ammonia or by allowing it 
to stand exposed to the air. When allowed to crystallize out slowly, 
calcium oxalate occurs in the form of well-defined, strongly refrac- 
tive octahedra, in which the principal axis of the crystals is placed 
at right angles to the plane of the microscope slide (Fig. 110). These 
are very characteristic. Other forms, however, are also quite com- 
monly observed, such as single and double dumb-bells, spheroids 
and prisms, etc. (Fig. 105). They are insoluble in ammonia and 
alcohol, almost insoluble in hot and cold water, and very slightly 
soluble in acetic acid, but dissolve with ease in the mineral acids. 

Fig. 110. 




Calcium oxalate crystals. 

When strongly heated, the salt is decomposed into calcium oxide, 
carbon dioxide, and carbon monoxide, according to the equation 

CaC 2 4 = CaO + C0 2 + CO. 

Tests for Oxalic Acid. — For the detection of calcium oxalate it 
is frequently only necessary to examine the sediment of the urine 
after standing for twenty-four to forty-eight hours. Xo oxalate 
crystals, however, may be found even when an abnormally large 
amount can be demonstrated by chemical methods. In such cases 
it is usually possible to bring about the crystallization of the salt by 
carefully neutralizing the urine with a little ammonia. Should this 
procedure not lead to the desired end, it is best to treat the urine 
with one-third its volume of 95 per cent, alcohol. The mixture is 
set aside for twenty-four to forty-eight hours, when the sediment is 



470 THE URINE. 

centrifugalized and examined with the microscope. This method, 
Baldwin states, represents a more delicate test for oxalic acid than 
the complicated methods of quantitative analysis which are available. 

Quantitative Estimation. — Heretofore the old method of Neu- 
bauer has been in general use, but it is at best unsatisfactory. It is 
still described at this place, as the more recent methods of Dunlop 
and Salkowski are as yet but little known. At the same time it 
must be admitted that these more modern procedures are likewise 
not free from objections, but they are nevertheless to be preferred to 
that of Neubauer. 

Neubauer's Method. — Principle. — The calcium oxalate in the urine 
is held in solution by the diacid sodium phosphate. If this is re- 
moved by means of calcium chloride and ammonia, the calcium 
oxalate is precipitated. By heating this strongly it is transformed 
into calcium oxide. 

As 56 parts by weight of calcium oxide correspond to 128 parts 
by weight of calcium oxalate, the amount of the latter can be 
readily calculated according to the equation : 56 :128 :: y :x ; and 
x = 2.2857 y, in which y indicates the amount of calcium oxide 
found in a given amount of urine, and x the corresponding amount 
of calcium oxalate. As 1 molecule of oxalic acid, moreover, corre- 
sponds to 1 molecule of calcium oxalate, the amount of the former 
can be found from that of the latter according to the equation : 
128 : 90 : : y : x ; and x — 0.703 y, in which y represents the amount 
of calcium oxalate found, and x the amount of the corresponding 
acid. 

Method. — A large amount of urine (600 to 1000 c.c.) is thy- 
molized, so as to guard against putrefactive processes, and is treated 
with an excess of calcium chloride solution and rendered strongly 
alkaline with ammonia. The diacid sodium phosphate which holds 
the oxalic acid in solution is thus removed. The precipitate of phos- 
phates is then carefully treated with an amount of acetic acid just 
sufficient to dissolve it, without filtering. As calcium oxalate is 
almost insoluble in acetic acid, it gradually separates out. To this 
end, the mixture is allowed to stand for twenty-four hours, the 
addition of the thymol preventing the development of bacteria. At 
the end of this time the calcium oxalate is filtered off through a 
small filter. It is washed with water and treated with a small 
amount of warm hydrochloric acid, any uric acid that may have 
separated out being left behind. The filtrate is further treated with 
a small amount of very dilute ammonia, so as to render the solution 
slightly alkaline. After standing for twenty-four hours the calcium 
oxalate will have separated out, and is collected upon a smaller filter, 
the weight of the ash in this being known. After washing with 
water the contents of the filter are dried and incinerated in a cruci- 
ble, heating strongly for about twenty minutes, whereby the oxalate 



CHEMISTRY OF THE URINE. 471 

is transformed into the oxide. From the weight of this the corre- 
sponding amount of oxalic acid is readily calculated according to 
directions given above. 

Dunlop's Method (slightly modified by Baldwin). — In this case the 
calcium oxalate is precipitated from an acid solution by means of 
alcohol, instead of from an alkaline solution by calcium chloride. 
The urine is thymolized, and if alkaline acidified with a trace of 
acetic acid. 

Five hundred c.c. of a well-mixed specimen of the collected urine 
of twenty-four hours are treated with 150 c.c. of over 90 per cent, 
alcohol, to precipitate the calcium oxalate. The mixture is set aside 
for forty-eight hours. It is then filtered, care being taken to insure 
the entire removal of the crystals from the beaker. The sediment 
is thoroughly washed with hot and cold water, and finally with 
dilute acetic acid (1 per cent, solution). The filter is placed in a 
small beaker and soaked in a small amount of dilute hydrochloric 
acid. It is then washed with hot water until the washings no 
longer give an acid reaction. The acid solution and washings 
are filtered, and the filtrate evaporated to about 20 c.c. This is 
treated with a very small amount of a solution of calcium chloride, 
to insure the presence of an excess of calcium. The solution is 
neutralized with ammonia, slightly acidified with acetic acid, and 
treated with strong alcohol, so that the mixture contains 50 per cent. 
After forty -eight hours the sediment is collected on a filter free from 
mineral ash, and is washed with cold water and dilute acetic acid 
until free from chlorides. The filter with its contents is then in- 
cinerated, first over a Bunsen burner, and afterward for five minutes 
in a blow-pipe flame. On cooling over sulphuric acid the ash is 
weighed; the result multiplied by 1.6 represents the amount of 
oxalic acid in the volume of urine examined. 

Salkowski's Method. — In the case of human urine of moderate 
concentration 500 c.c. of the non-filtered urine are evaporated to 
about one-third. On cooling, the liquid is acidified with 20 c.c. of 
hydrochloric acid (sp. gr. 1.12), and extracted three times with new 
portions of 200 c.c. each of a mixture of 9 to 10 volumes of ether 
and 1 volume of alcohol. The ethereal extracts, which contain 
the liberated oxalic acid, are carefully separated from the urine 
and filtered through a dry filter. The ether is distilled off; the re- 
maining alcoholic solution, which still contains a little ether, is placed 
in a deep evaporating-dish, diluted with 10 to 15 c.c. of water, and 
evaporated on a water-bath. The resulting milky fluid is concen- 
trated, more water being added if necessary, until it becomes clear 
and a gummy material separates out. On cooling, the liquid, which 
should measure about 20 c.c, is passed through a small filter. This 
is washed once or twice with a little water, when filtrate and washings 
are rendered slightly alkaline with ammonia, treated with 1 to 2 c.c. 



472 THE URINE. 

of a 10 per cent, solution of calcium chloride, and acidified with 
dilute acetic acid. The reaction should be distinctly acid, but an 
excess should be avoided. An indication that a sufficient amount 
has been added is afforded by the dissolution of the precipitate of 
phosphates, which occurs after the addition of the calcium chloride 
solution. After standing for twenty-four hours, or still better forty- 
eight hours, the calcium oxalate that has separated out is collected 
on a filter free from ash, washed with hot and cold water, dried, and 
incinerated as usual (see above). The resulting weight, multi- 
plied by 1.6 indicates the corresponding amount of oxalic acid in 
grammes. 

Literature. — P. Fiirbringer, " Zur Oxalsiiureausscheidung durch d. Harn," 
Deutsch. Arch. f. klin. Med., 1876, vol. xviii. p. 143. J. C. Dunlop, " The Elimination 
of Oxalic Acid in the Urine," etc., Jour. Path, and Bact., 1896 (an historical review 
of the subject of oxaluria is here also given). H. Baldwin, " An Experimental Study 
of Oxaluria," Jour. Exper. Med., vol. v. p. 27. E. Salkowski, Berlin, klin. Woch., 1900, 
p. 434 ; and Zeit. f. physiol. Chem., vol. xxix. p. 437. E. Harnack, " Ueber Indican- 
urie in Folge von Oxalsaurewirkung," Zeit. f. physiol. Chem., 1900, vol. xxix. p. 205. 

ALBUMINS. 

The albumins which may be met with in the urine are serum- 
albumin, serum-globulin, albumoses (peptones), the albumin of 
Bence Jones, haemoglobin, nucleo-albumin, fibrin, histon, and nucleo- 
histon. Of these, serum-albumin is the most important from a 
clinical standpoint. 

Serum-albumin. — The question whether or not serum-albumin 
occurs normally in the urine — i. e., under strictly physiological con- 
ditions — has been much disputed. It is claimed by some that traces 
may be temporarily met with in apparently healthy individuals after 
severe muscular exercise, cold baths, mental labor, severe emotions, 
during menstruation, digestion, etc. This so-called physiological albu- 
minuria mostly occurs in young adults, and is usually, if not always, 
of brief duration. The urine, it is claimed, is otherwise normal — 
i. e. y of normal amount, appearance, specific gravity, and composi- 
tion, and free from abnormal morphological constituents, such as 
casts, red corpuscles, leucocytes, and epithelial cells. 1 

The existence of a physiological albuminuria, on the other hand, 
is denied, and the occurrence of serum-albumin at least regarded as 
pathological in every case. I have never been able to convince my- 
self of the occurrence of serum-albumiu in the urine under strictly 
physiological conditions, and it has been pointed out elsewhere 
that severe muscular and mental labor, severe mental emotions, 
cold baths, etc., can hardly be regarded as physiological stimuli for 
all persons. The albuminuria, so often observed during the first 
days of life, at which time sediments of uric acid and urates, mucus, 
epithelial cells from the different portions of the urinary tract, and 

1 C. E. Simon, " Functional Albuminuria," N. Y. Med. Jour., 1895, p. 330. 



. ALBUMINS. 473 

even casts may also be seen — i. e., constituents which in adults 
would rightly be regarded as abnormal — has also been brought for- 
ward in support of the theory of a physiological albuminuria. 
There can be no doubt, however, that this form of albuminuria is 
referable to the profound changes that take place in the circulatory 
system after birth, and to some extent perhaps also to the well- 
known uric-acid infarctions so frequently seen in the kidneys of the 
newly born, so that it would probably be better and more in accord 
with the teachings of pathology to regard this form of albuminuria 
also as abnormal. 1 

The more closely the subject of the so-called physiological albu- 
minuria is studied the more improbable does its physiological nature 
appear, and a more detailed study of the metabolic processes, it may 
be confidently asserted, will ultimately lead to the conclusion that 
the 'presence of albumin in every case is a pathological phenomenon. 

The association of an increased elimination of urea and uric acid 
with albuminuria in apparently healthy individuals was noted twenty- 
five years ago, but received comparatively little attention. More 
recently, Da Costa 2 has pointed out the existence of albuminuria 
associated with lithuria and oxaluria. Personal observations have 
led me to look upon this form of albuminuria as of common occur- 
rence, and while in almost every case the albumin can be caused to 
disappear from the urine by proper diet and exercise, there can be 
no doubt that, if neglected, granular atrophy may ultimately result. 

An albuminuria may at times be observed in anaemic children 
and adolescents, and particularly in masturbating boys of the mouth- 
breathing type, but can hardly be regarded as physiological. The 
same may be said of the albuminuria of pregnancy and parturition. 

As regards the action of cold baths, Pem-Picci 3 reports that 
albuminuria may be considered a constant phenomenon after cold 
baths, but that different subjects react differently under the same 
conditions. Those which show albuminuria more readily are, as a 
rule, the less robust and thinner individuals, such as are most sensi- 
tive to cold. The limits of temperature 'necessary to produce the 
phenomenon are from 12° to 13° C, when the immersion is not 
longer than three minutes. If the temperature be from 15° to 20° C, 
the albumin appears only after fifteen minutes' immersion. Above 
this temperature albuminuria does not occur, even if the bath lasts 
much longer. The colder the bath the more rapid the appearance 
of albumin. The degree of albuminuria is always slight, and even 
in the more marked cases rarely exceeds 0.25 pro mille. The 
sediment, according to Rem-Picci, occasionally shows a few hyaline 
casts, and often crystals of calcium oxalate. 

1 L. Landi, L'albuminuria nel parto, Morgagni, 1890, vol. xxxii. 

2 Da Costa, "The Albuminuria and Bright's Disease of Uric Acid and Oxalic 
Acid," Am. Jour. Med. Sci., 1895. 

3 Kem-Picci, "On Albuminuria after Cold Baths," II Policlinico, 1901, vol. viii. 
p. 389. 



474 THE URINE. 

The course which may be taken by these various forms of what 
should be termed functional albuminuria, in which the amount of 
albumin rarely exceeds 0.1 per cent., is very interesting. The elimi- 
nation of albumin may thus be quite transitory on the one hand, as 
when following severe muscular exercise, cold baths, and the like. 
It may, however, also last for several days, or even weeks, and be 
followed by a disappearance of the albumin for a variable length of 
time, and again by its reappearance and continuance for days and 
weeks. The term intermittent albuminuria 1 has been applied to this 
latter type. At times the albuminuria may follow a definite course, 
disappearing and reappearing with such regularity that it has not 
improperly been styled cyclic albuminuria. 2 In this form the albu- 
min generally disappears from the urine during the night or during 
prolonged rest in bed, and reappears during the day, the erect 
posture apparently favoring its reappearance ; the term postural cr 
orthostatic albuminuria has hence also been suggested for this form. 
Oswald, who made a careful study of cyclic albuminuria in RiegePs 
clinic, regards its occurrence as distinctly pathological, and as indi- 
cating the existence of nephritis. Remembering the importance of 
the subject, it may not be out of place to enumerate the reasons 
which led Oswald 3 to this conclusion : 

1. The patients generally come to the physician complaining of 
certain definite symptoms which are similar to those noted in cases 
of true nephritis. At times, however, no complaints are made, be- 
cause the patients have reasons for concealing them (as in examina- 
tions for life-insurance), or because they are temporarily absent. 

2. The subjective complaints, as well as the anaemia so frequently 
observed in such cases, generally disappear, together with the albu- 
min, under suitable treatment, and reappear when the anaemia again 
becomes marked. 

3. In many, a history of an antecedent nephritis the result of 
scarlatina or diphtheria may be obtained, as in three cases of Heub- 
ner, in fourteen cases out of twenty described by Johnson, etc. In 
some also a direct transition from an acute nephritis to the cyclic 
form of albuminuria has been noted. Where this was not possible 
the history of an acute infectious disease or an angina that had been 
overlooked in the clinical history must be regarded as a possible cause. 

4. The absence of morphological elements, especially tube-casts, 
does not exclude a nephritis. A large number of cases, moreover, 
have recently been observed in which casts were repeatedly found. 

5. A cyclic albuminuria may be observed in many cases of 
chronic nephritis. 

i Bull, Berlin, klin. Woch., 1886, vol. xxiii. p. 717. Mareau, Eev. de med., 1886, 
vol. vi. p. 855. Klemperer, Zeit. f. klin. Med., 1887, vol. xii. p. 168. 

2 A. Keller, Beitrage z. Kenntniss d. cyklischen Albumin urie, Diss., Breslau, 1896. 

3 K. Oswald, •'Cyklische Albuniinurie u. Nephritis," Zeit. f. klin. Med., vol. xxvii. 
p. 73. 



ALBUMINS. 475 

6. Marked organic abnormalities (such as heart-lesions) need not 
be demonstrable, as they may be absent for a long period of time or 
may be unrecognizable. 

Senator's l statement, that the existence of a physiological albu- 
minuria is proved by the fact that the morphological constituents of 
the primitive nubecula contain albumin, requires no further criticism, 
and should be regarded as a misconstruction of the main point at 
i ssue — a mere sophism ; and Posner' s 2 observations, in view of the 
researches of Malfatti, 3 which tend to show that the body obtained 
by Posner was not serum-albumin, but a nucleo-albumin, may now 
be regarded as erroneous. 

In conclusion, it may be safely asserted that a transitory, intermit- 
tent, and cyclic albuminuria is not infrequently observed in apparently 
healthy individuals, but that the facts so far brought forward do not 
warrant the assumption that such forms of albuminuria are physio- 
logical. 41 The occurrence of such albuminuria unquestionably demon- 
strates a certain insufficiency of the renal epithelium, and I am much 
in favor, as Martius has proposed, of discarding the term physiological 
albuminuria altogether, and to speak of these various forms collec- 
tively as constitutional albuminuria. 

It would lead too far to enter into a detailed consideration of the 
various causes that have from time to time been suggested as an ex- 
planation of the fact that albumin does not occur in the urine under 
normal conditions. There can be no doubt, however, that the integ- 
rity of the epithelial lining of the glomeruli and the convoluted 
tubules must be regarded as the principal factor which prevents the 
albumin of the blood from passing into the urine. When the readi- 
ness with which the glandular structures of the kidney respond to 
any abnormal stimulation is considered, it is easily understood how 
an albuminuria may be produced in many different ways. Aside 
from acute and chronic inflammatory processes in the widest sense 
of the word, an albuminuria may be the result of circulatory dis- 
turbances in the kidneys of whatever kind — i. e., the result of 
anaemia as well as of hyperemia. In many and perhaps the 
majority of cases in which what Bamberger 5 terms a hematogenous 
albuminuria occurs, we have direct evidence of the existence of cir- 
culatory disturbances, as in cases of uncompensated valvular lesions, 
weak heart, emphysema, hepatic cirrhosis, etc. In other cases, how- 
ever, the existence of such disturbance can only be surmised, and the 

1 Senator, Die Albuminurie, Hirschwald, Berlin, 1882. 

2 C. Posner, Berlin, klin. Woch., 1885, vol. xxii. p. 654 ; Virchow's Archiv, 1886, 
vol. civ. p. 497 ; Arch. f. Anat. u. Physiol., 1888. 

3 Malfatti, Internat. Centralbl. f. d. Physiol, u. Pathol, d. Harn- u. Sexualorgane, 
1889, vol. i. p. 266. 

4 v. Noorden, Deutsch. Arch. f. klin. Med., vol. xxxviii. pp. 3 and 205. Leube, 
Zeit. f. klin. Med., 1887, vol. xiii. p. 1. Winternitz, Zeit. f. physiol. Chem., 1891, vol. 
xv. p. 189. C. E. Simon, loc. cit. 

5 v. Bamberger, Wien. med. Woch., 1881, pp. 145 and 177. 



476 THE URINE. 

question, whether or not the albuminuria observed in the various 
infectious diseases, for example, is referable to circulatory abnormali- 
ties or to a direct irritative action of microbic poisons upon the 
renal parenchyma, must still remain open. 

From personal studies in connection with the functional albu- 
minuria of Da Costa, it seems not unlikely that in many cases in 
which obscure circulatory disturbances are supposed to exist and are 
held responsible for an existing albuminuria, this is referable rather 
to the strain thrown upon the kidneys by the continued elimination 
of abnormally large quantities of organic material, the quantity of 
water being at the same time proportionately small. 

If it is remembered, furthermore, that injuries affecting certain 
portions of the brain are followed by albuminuria, and that this 
may be artificially produced by a piqure, analogous to the glucosuric 
piqure of C. Bernard, still another factor is given which may pos- 
sibly enter into the causation of albuminuria. 

Obstruction to the outflow of urine from the kidneys has also 
been experimentally shown to lead to albuminuria, an observation 
with which clinical experience is in perfect accord. 

In patients actually in labor albuminuria is common, and supposedly 
due to increased blood-pressure in the kidneys caused by uterine 
contractions and the general disturbance of the circulation. The 
relative frequency of its occurrence is a matter of dispute, however, 
and widely differing statements are made by different observers, 
ranging from 15 to 20 per cent. (Petit, Winckel) to 99 and 100 per 
cent. (Trautenroth, Pajikull). 

As regards the occurrence of albuminuria in pregnancy the results 
of different observers likewise differ, viz., from 1 to 50 per cent. 
In the last months of pregnancy Zangemeister l found albumin in 10 
per cent, of the cases examined, and if repeated examinations were 
made positive results were obtained persistently during the last three 
months in 40 per cent. The albuminuria is supposedly referable to 
some metabolic disturbance and impaired excretion by the kidneys. 

Finally, an abnormal composition of the blood may at times cause 
the albuminuria. 

In passing on to a more detailed study of the various pathological 
conditions in which an elimination of albumin may be noted, an 
attempt will be made to classify the various forms of albuminuria 
in accordance with the more general considerations set forth above. 
It should be remembered, however, as already indicated, that it may 
be very difficult, if not impossible, to assign one single cause to 
a given clinical case, as several factors may at the same time be 
operative in the production of the albuminuria. 

1. Functional Albuminuria. — Under this heading may be 
comprised the various forms of " physiological " albuminuria, which 
have already been considered. 

1 Zangemeister, Arch. f. Gyn., 1902, vol. lxvi. Heft 2. 



ALBUMINS. 477 

2. The albuminuria associated with organic diseases 
of the kidneys, viz., acute and chronic nephritis, renal arterio- 
sclerosis, amyloid degeneration of the kidneys. 1 

In acute nephritis, albuminuria, usually of great intensity, is a 
constant and most important symptom. The amount eliminated 
is generally proportionate to the intensity of the disease, but varies 
within fairly wide limits, generally from 0.3 to 1 per cent., corre- 
sponding to a daily excretion of from 5 to 8 grammes. Much larger 
quantities, it is true, are at times excreted, but it may be definitely 
stated that the daily loss of albumin seldom exceeds 20 grammes. 

In chronic parenchymatous nephritis the elimination of albumin 
is likewise constant, and the amount excreted in severe cases may 
even exceed that observed in the acute form. An elimination of 
from 15 to 30 grammes, viz., 1.5 to 3 per cent, by weight, is 
frequently observed. 

In the ordinary form of chronic interstitial nephritis the elimina- 
tion of albumin is, as a general rule, slight, and rarely amounts to 
more than 2 to 5 grammes pro die. At the same time it is not 
unusual to meet with an apparent absence of albumin if the more 
common tests (see below) are employed. If it is remembered that 
very often the diagnosis of the disease is dependent upon the demon- 
stration of the presence or absence of albumin, the necessity of fre- 
quent examinations and the employment of more delicate tests, par- 
ticularly of the trichloracetic acid test, as well as of a microscopical 
examination, is at once apparent. This is even of greater impor- 
tance in the renal arteriosclerosis of Senator, in which albumin by 
the ordinary tests is probably not demonstrable in the majority of 
cases, and in which even the trichloracetic acid test may not be of 
service, and casts are absent. 

Amyloid degeneration of the kidneys, in the absence of inflamma- 
tory processes, is accompanied by a condition of the urine closely 
resembling that observed in the ordinary form of chronic interstitial 
nephritis. A total absence of albumin, however, is less frequently 
noted, while an amount varying between 1 and 2 per cent, is not 
uncommon. It will be shown later on that in this condition con- 
siderable amounts of serum-globulin are excreted in addition to 
the serum-albumin; larger amounts, in fact, than are generally 
observed in this form of chronic renal disease, so that Senator sug- 
gests that such a relation, in the absence of an acute nephritis, or 
an acute exacerbation of a chronic nephritis, may be of a certain 
diagnostic value. 

3. Febrile Albuminuria. 2 — That albuminuria may occur in 
almost any one of the various febrile diseases is a well-known fact, 

1 Senator, loc. cit. 

2 Leyden, Zeit. f. klin. Med., 1881, vol. iii. p. 161. H. Lorenz, Wien. klin. Woch., 
1888, vol. i. p. 119. 



478 THE URINE. 

but it is important to remember that, while such an albuminuria 
may at times be referable to a true nephritis developing in the course 
of or during convalescence from an acute febrile disease, such is the 
exception, and not the rule. Under this heading, only that form 
will be considered which is not associated with distinct changes 
affecting the renal parenchyma, and which generally appears during 
the height of the disease only, and disappears with a return of the 
temperature to normal. As has been mentioned, it is often 
difficult, if not impossible, to assign a definite cause for an albu- 
minuria of this character, and in all probability several factors are 
in operation at the same time. In the beginning of the disease, 
when the blood-pressure, as a rule, is increased, the albuminuria 
may be referable to an ischsemia of the kidneys, as the increased 
pressure in fever, according to Cohnheim and Mendelson, is largely 
referable to spasm of the arterioles. Later on, or in the begin- 
ning of cases in which especially severe intoxication exists, the 
blood-pressure may be subnormal, and the albuminuria be due to 
this cause — i. e., a hyperaemic condition of the kidneys. As a mat- 
ter of fact, it has been experimentally demonstrated that both anaemia 
and hyperemia of the kidney structure may lead to albuminuria. 
On the other hand, it is not unlikely that the strain thrown 
upon the kidneys by an excessive elimination of organic material, 
in the absence of a correspondingly large quantity of water, may 
produce albuminuria. I have repeatedly seen the functional 
albuminuria of the type described by Da Costa disappear during 
the administration of a diet relatively poor in nitrogen, while an 
increased diuresis was at the same time effected by the consumption 
of large amounts of water. 

In those grave cases of typhoid fever, furthermore, which are 
characterized by high fever and pronounced nervous symptoms it 
would appear quite likely that the albuminuria, which in these cases 
is particularly marked, is referable to a direct influence upon the 
central nervous system, and in some cases, at least, also dependent 
upon an irritant action upon the renal epithelium on the part of the 
microbic poisons circulating in the blood. The character of the albu- 
minuria will largely depend upon the intensity of the intoxication ; 
in other words, upon the amount of bacterial poison present at any 
one time in the blood. 

Notwithstanding statements to the contrary, albuminuria may be 
regarded as a constant symptom of typhoid fever, as has been defi- 
nitely demonstrated by Gubler and Robin. It is difficult to say why 
other observers have found albumin in only a comparatively small 
percentage of cases, but it is not unlikely that this is owing to a lack 
of uniformity in methods, it being presupposed also that questions 
of this kind can only be decided by daily examinations. According 
to Robin, the trace of albumin which is at times observed during 



ALBUMINS. 479 

the first week of the disease is an albumose, while later on serum- 
albumin is constantly found ; the amount increases with the inten- 
sity of the morbid process, and the highest figures are reached in fatal 
cases. The more severe the disease the earlier does albumin appear 
in the urine, it being remembered, however, that reference is had 
only to those cases in which distinct renal changes are not demon- 
strable. Toward the termination of the fastigium the amount of 
albumin generally undergoes a certain diminution, and may even 
disappear entirely. This diminution, however, is only temporary, 
and in severe eases the albumin again increases in amount during 
the period of great variations in the temperature. In light cases 
an increased elimination also takes place at this stage, but is soon 
followed by a decrease, after which only traces can be demonstrated. 
In some cases it disappears entirely, but it is rare, according to Robin, 
to meet with cases in which at least a trace does not reappear during 
convalescence. 

In light cases the albuminuria rarely persists longer than the fifth 
or eighth day of convalescence, and Robin even goes so far as to say 
that a relapse may be anticipated if the albuminuria does not disap- 
pear at that time. A limited number of personal observations have 
borne out the correctness of this view, and in one case in which a 
relapse occurred so late as the fifteenth day of convalescence traces 
of albumin could be demonstrated during the entire period. In 
severe cases, on the other hand, the albumin persists for a variable 
length of time, and rarely disappears before the tenth day of con- 
valescence. At times an increase is seen during convalescence when 
traces only have previously been observed. It is this form which 
the French generally speak of as colliquative albuminuria. While 
this is principally observed in typhoid fever, it is not unusual 
to meet with it during convalescence from various other acute 
diseases. Care must be taken not to confound the albuminuria so 
frequently seen during convalescence from typhoid fever, referable 
to a pyelitis, with the form just described. 

From the following summary, constructed from data given in 
Robin's l monograph on the urine of typhoid fever and other acute 
infectious diseases which may be associated with a typhoid condition, 
an idea may be formed of the occurrence of albuminuria, as well as 
of its degree of intensity in these diseases : 

Acute miliary tuberculosis : albumin is much less frequent than 
in typhoid fever ; when present, it is rarely found in the abundance 
so characteristic of the fatal cases of the latter disease. 

Pneumonia : albumin is as uniformly present as in typhoid fever, 
and at times very abundant. 

Grippe : albumin is infrequent ; present in about 20 per cent, of 
the cases, and only in traces. 

1 A. Robin, Urologie clinique de la fievre typhoide, Paris, 1877. 



480 THE URINE. 

Herpetic fever : albumin never present in large amounts. 

Embarras gastrique : albumin rarely present. 

Adynamic enteritis of adults : albumin almost always present, but 
usually only in traces. 

Cerebrospinal meningitis : albumin in fairly large amounts. 

Vegetative endocarditis : albumin very abundant in about 14 per 
cent., evident in 44 per cent., and traces in 42 per cent, of all cases. 

Acute articular rheumatism : albumin present in about 40 per 
cent. 

Rubeola : albumin usually absent in light cases, but present in 
the more severe and complicated forms. 

Intermittent fever : albumin variable. 

In a series of 799 cases of pneumonia reported from the Boston 
City Hospital, 1 albumin was found in 624 — i. <?., in 78 per cent. It 
was noted that the death-rate bore a direct ratio to the amount of 
albumin in the urine. 

In smallpox a trace of albumin is practically constant. Some- 
what larger amounts are found in about 30 per cent, of all cases. 
The albuminuria is most marked during the eruptive stage and then 
rapidly diminishes in intensity. More rarely it reaches its maximum 
during the suppurative fever stage, or during convalescence. 2 

As the result of the examination of a large number of cases of 
plague Corthorn 3 arrived at the conclusion that no albumin is found 
in only 14 per cent, of all cases. In cases ending in recovery the 
albuminuria never occurred later than the fourth day. 

In conclusion, it may be said that practically every acute febrile 
disease, even simple follicular tonsillitis, may be accompanied by 
albuminuria in the absence of definite changes affecting the renal 
parenchyma. Its occurrence in an individual case is probably 
dependent to a very large degree upon the intensity of the intoxica- 
tion. While it is generally an easy matter to distinguish between 
this form of albuminuria and that associated with distinct organic 
changes in the kidneys, considerable difficulty may at times be 
experienced ; this question will be dealt with later on. 

4. Albuminuria referable to Circulatory Disturbances. 4 
— To this class belongs the albuminuria so frequently observed in 
cardiac insufficiency referable to valvular lesions, degeneration of the 
heart-muscle from whatever cause, disease of the coronary arteries, 
etc., as well as in cases of impeded pulmonary circulation affecting 
the general circulation through the right heart, and, finally, in con- 
ditions associated with local circulatory disturbances, such as com- 

1 Sears and Larrabee, Med. and Surg. Rep. of the Boston City Hospital, 12th Series, 
Dec, 1901. 

2 Arnaud, " Albuminuric et lesions des reins dans la variole," Rev. d. Med., 1898, 
vol. xviii. p. 392. 

3 Corthorn, " Albuminuria in Plague," Brit. Med. Jour., Sept. 14, 1901. 

4 Senator, loc. cit. 






ALBUMINS. 481 

pression of the renal veins by a pregnant uterus, tumors, etc. It 
has been pointed out that febrile albuminuria also may, to a certain 
extent at least, be referable to such causes — i. e. y an ischsemia or 
hyperemia of the kidneys produced by an increased or diminished 
blood-pressure. The albuminuria observed in cases of cholera 
infantum, the simpler forms of intestinal catarrh, and in cholera 
Asiatica particularly, are undoubtedly dependent upon such causes. 
The occurrence of albuminuria after cold baths, as stated above, is 
regarded by many as a " physiological " phenomenon ; but this view 
should be rejected, as there can be little doubt that this form is also 
referable to circulatory disturbances. The quantity of albumin 
found under these circumstances varies considerably, but rarely 
exceeds 0.1-0.2 per cent, unless the disease has advanced to a stage 
where distinct changes in the renal parenchyma have resulted. 

5. Albuminuria referable to an Impeded Outflow of 
Urine. — Clinically, albuminuria referable primarily to an impeded 
outflow of urine from the kidneys is probably of more frequent 
occurrence than is generally supposed, and especially in women, in 
whom Kelly and others have demonstrated the frequent existence 
of ureteral stenoses. A complete blocking of the excretory duct, 
on the other hand, is rarely seen, but may be caused by the impac- 
tion of a renal calculus, the pressure of a tumor, or following cer- 
tain gynaecological operations in which the ureter is accidentally 
caught in a suture, etc. It has also been suggested that the albu- 
minuria of pregnancy may be due to compression of a ureter, but 
it is more likely that other factors are here at play, such as com- 
pression of the renal arteries and veins. 

6. Albuminuria of H^mic Origin. 1 — It was formerly sup- 
posed that Bright' s disease was dependent upon certain abnormalities 
of the blood, and as a matter of fact this view has not only never 
been disproved, but is actually gaining ground from day to day. 
According to Semmola, Bright' s disease is primarily due to an 
abnormal power of diffusion on the part of the albumins of the 
blood, which are eliminated by the kidneys as waste material. As 
a result of the excessive amount of work thus done definite renal 
changes are finally produced. According to his theory, then, the 
albuminuria is the primary factor in the causation of nephritis. 
Should this hypothesis hold good, Senator is correct in asserting that 
an albuminuria of functioual origin, so to speak, must precede the 
occurrence of the nephritis proper. He, however, doubts the occur- 
rence of a prenephritic albuminuria ; but others have noted the occur- 
rence of definite renal changes which manifestly followed an appar- 
ently functional albuminuria (Da Costa). Further researches in this 
direction are urgently needed, and Semruola's view can at present only 
be regarded as an hypothesis. But even if such blood-changes as 

1 v. Bamberger, loc. cit. 
31 



482 THE URINE. 

those which Semmola suggests should not exist, there can be little doubt 
that true nephritis is dependent upon an acute or chronic dyscrasia 
of the blood, either in the sense of an abnormal mixture of the nor- 
mal elements or of the presence of abnormal constituents, and not- 
ably of poisons. The same considerations undoubtedly also apply 
to various other forms of albuminuria, in so far as these are not the 
direct result of circulatory disturbances. 

Clinically, albuminuria of hsemic origin is observed in various 
diseases of the blood, such as purpura, scurvy, leukaemia, pernicious 
anaemia, as also in cases of poisoning with lead and mercury, in 
syphilis, jaundice, diabetes, following the inhalation of ether and 
chloroform, etc. The albuminuria associated with an excessive 
elimination of uric acid and oxalic acid, and, according to personal 
observations, with an excessive elimination of organic material in 
general, notably of urea; probably also belongs to this class. 

7. Toxic Albuminuria. — It has already been stated that the 
albuminuria of acute febrile diseases may, to a certain extent, be 
referable to a direct irritant action on the part of bacterial poisons 
upon the renal parenchyma. Poisoning with cantharides, mustard, 
oil of turpentine, potassium nitrate, carbolic acid, salicylic acid, tar, 
iodine, petroleum, phosphorus, arsenic, lead, antimony, alcohol, and 
mineral acids produces albuminuria. In all probability, however, 
the albuminuria here observed is referable not only to a direct irri- 
tant action upon the glandular epithelium of the kidneys, but also 
to circulatory disturbances. 

8. Neurotic Albuminuria. — It is claimed by some that albu- 
min, usually in small amounts, is eliminated in epilepsy after every 
attack, while others either deny its occurrence under such conditions 
or regard it as exceptional. In a number of cases in which I had 
occasion to examine urine voided after an attack albumin was usually 
absent. It should be stated, however, that the seizures in these 
cases were comparatively slight, and that unfortunately an exam- 
ination for semen was not made in those urines in which traces of 
albumin were demonstrated. An examination of the urine voided by 
a patient, after having been in the epileptic state for more than forty- 
eight hours, showed the presence of a small amount of albumin 
associated with an enormous elimination of uric acid, as well as a 
large excess of urea. Semen was absent. 1 Nothnagel states that he 
could not demonstrate any regularity in the appearance of albumin. 
In some of his cases with major attacks there was no albumin ; in 
others it appeared after every attack ; in still others it was some- 
times present and at other times absent (in the same individual). 
At times it was found after a minor attack and was absent after a 
major attack (also in the same individual). 

Other observers have obtained similar results, so that we may 

1 M. Huppert, Virchow's Archiv, 1874, vol. lix. p. 305, 



ALBUMINS. 483 

conclude that albuminuria following epileptic seizures is rather the 
exception than the rule. When it does occur, its significance is 
essentially the expression of a certain grade of cyanosis during the 
attacks. 1 

A transient albuminuria has also been noted in cases of progressive 
paralysis, mania, tetanus, delirium tremens, apoplexy, migraine, 
Basedow's disease, brain tumor, etc. 

Although albuminuria may apparently be produced artificially by 
injuries affecting a certain area in the floor of the fourth ventricle, 
analogous to the production of glucosuria (see Glucosuria), it would 
probably be going too far to assume the existence of a certain spe- 
cific centre, stimulation of which causes the appearance of albumin 
in the urine. While the influence of the nervous system in prevent- 
ing the passage of albumin through the glomeruli under normal 
conditions is undoubted, it would appear more likely that the albu- 
minuria following injuries to the central nervous system is referable 
to circulatory disturbances in the kidneys secondary to lesions of 
the brain, and especially of the medulla. The albuminuria observed 
in certain neurotic individuals, on the other hand, is probably more fre- 
quently associated with metabolic abnormalities, and is of hsemic origin. 

9. A digestive albuminuria has also been described. 2 It may 
follow the ingestion of excessive amounts of cheese, eggs — particu- 
larly when taken raw — beef, etc. Specially interesting is the form 
which follows the ingestion of excessive amounts of egg albumin. 
Ordinarily the consumption of a moderate amount of such albumin 
does not lead to albuminuria, while in cases of nephritis an already 
existing albuminuria is increased. But it has also been noted that 
even in individuals with apparently healthy kidneys, the ingestion 
of an excessive amount of egg albumin may call forth albuminuria, 
and it is possible in both cases to demonstrate the presence in the 
urine of both egg albumin and blood albumin. 

To examine into this question the individual is given from four to 
eight raw eggs on an empty stomach in the morning for two to four 
days. His diet otherwise is as usual. The urine is collected in 
intervals of from two to three hours. If the ingestion of such an 
amount of egg albumin leads to albuminuria, this usually occurs 
after about four hours, and reaches its maximum intensity two 
hours later. Casts are not found (Jnouye). 

The albuminuria in question, so far as the egg albuminuria goes, 
is undoubtedly owing to the fact that a certain amount of egg albu- 
min is absorbed as such from the gastro-intestinal canal and is subse- 
quently eliminated as foreign material. In what manner, however, 

1 Nothnagel, Ziemssen's Handbuch, 1877, vol. xii. p. 179. Binswanger, NothnagePs 
spec. Pathol, u. Therap., vol. xii. p. 235 (literature). 

2 Ascoli, "Ueber d. Mechanismus d. Albuminuric durcb Eiereiweiss," Munch, med. 
Woch., 1902, No. 10. Jnouye, "Ueber alimentare Albuminuric," Deutsch. Arch. f. 
klin. Med., 1902, vol. lxxv. p. 378, 



484 THE URINE. 

the egg albuminuria may be responsible for the accompanying 
serum albuminuria is more difficult to explain. 

Of the albuminuria which follows excessive indulgence in cheese 
and beef but little is known. Bearing in mind that the albu- 
minuria very often follows the ingestion of such articles almost 
immediately, and before they have become absorbed, it is hardly 
justifiable to refer this form to the existence of a hyperalbuminosis. 
It would appear more rational, as Senator has suggested, to think 
of reflex vasomotor or trophic changes affecting the kidneys ; while 
in other cases, in which the albuminuria does not follow the inges- 
tion of such articles of food immediately, it is quite probable that 
is may be dependent upon certain metabolic abnormalities affecting 
the normal composition of the blood. 

In the account thus given of the occurrence of albuminuria and 
its possible causes, reference has been had to only a purely renal albu- 
minuria. It should be remembered, however, that the origin of the 
albumin may often be extremely difficult to determine, as albuminous 
material, such as blood and pus, may become mixed beyond the 
glandular portion of the kidneys with what would otherwise have 
been a perfectly normal urine, and that such an admixture may take 
place not only in the ureters, the bladder, and the urethra, but even 
in the pelvis of the kidney. 

The term accidental albuminuria is applied to a condition in which 
albuminous material becomes mixed with a urine beyond the kidneys, 
as in cases of cystitis and urethritis, or whenever semen has entered 
the urine while the renal urine proper is free from albumin. An ad- 
mixture of pus, blood, lymph, or chyle may, however, also occur in the 
kidneys, when the albuminuria is termed accidental renal albuminuria, 
an example of which is frequently seen in the slight degree of albu- 
minuria referable to pyelitis during convalescence from typhoid 
fever. By a mixed albuminuria and a mixed renal albuminuria, on 
the other hand, we are to understand conditions in which the source 
of the albumin is twofold, renal and extrarenal in the first instance, 
parenchymal and extraparenchymal in the second, examples being the 
albuminuria of cystitis combined with nephritis and pyelonephritis, 
respectively. 

It is manifest, of course, that in every instance in which albumin 
is found in the urine its origin should be ascertained. While this 
question is usually readily decided by a microscopical examination 
of the urine, considerable difficulty may occasionally be experienced. 
It is a well-known fact that in the urine of women a trace of albu- 
min may frequently be detected, which is not due to any lesion of 
the urinary organs, but to an admixture of vaginal discharge, of 
blood during the process of menstruation, and, in married women, 
of semen. Whenever, therefore, doubt is felt as to the origin of the 
albumin, the specimen for examination should be obtained by the 



ALBUMINS. 485 

catheter, care being taken previously to cleanse the vulva. In men 
albumin may be referable to a gonorrhoea! urethritis. In such cases 
it is well to let the patient flush out his urethra first, and to make 
use for examination of the portion last voided. Very often, how- 
ever, the conditions are more complex, it being uncertain whether 
the albumin is referable to the presence of pus only, or whether its 
origin is in the renal parenchyma. In such cases, as in cystitis, 
pyelonephritis, etc., a careful microscopical examination and enumer- 
ation of the pus-corpuscles with the Thoma-Zeiss instrument are 
called for, and will in the majority of instances decide the question. 
Generally speaking, the amount of albumin found in uncomplicated 
cases of cystitis does not exceed 0.15 per cent., while in cases of 
pyelitis of the same intensity the amount of albumin is from two to 
three times as large. 

Of late, attention has repeatedly been drawn to the occasional 
presence in the urine of an albuminous body which is soluble in 
acetic acid, and which Patein regards as a modification of common 
serum-albumin. It has thus far been observed in only eight cases, 
viz., twice in chronic nephritis, three times in eclampsia, once in a 
cystic kidney, once in tonsillitis following an injection of diphtheria 
antitoxin, and once in a pregnant woman in whom typhoid fever, 
developed. I should suggest that the substance be spoken of as 
Patein's albumin l until its chemical identity has been established. 
The term aceto-soluble albumin is, of course, likewise admissible. 

So far as the amount of albumin which may be eliminated in the 
twenty-four hours is concerned, an excretion of less than 2 grammes 
may be regarded as insignificant, 6 to 8 grammes as a moderate 
amount, and 10 to 12 grammes or more as excessive. An excretion 
of 20 to 30 grammes is exceptional. 

Serum-globulin.- — It has been pointed out that in cases of amyloid 
degeneration of the kidneys serum-globulin is found in the urine 
together with serum-albumin in large amounts, and, according to 
Senator, a ratio between the two albumins of 1 : 0.8 : 1.4 may be 
regarded as a fairly -constant symptom of the disease, and is of diag- 
nostic importance. There seems to be no doubt, however, that 
serum-globulin occurs in the urine, although in much smaller quan- 
tities than in the disease mentioned, whenever serum-albumin is 
eliminated. 2 

A most remarkable instance of globulinuria has been recorded 
by Noel Paton, 3 in which the globulin separated out in crystalline 
form and was found in extraordinarily large quantity, amounting 
on one day to 70 grammes. 

1 Patein, "Aceto-soluble Albumin in the Urine," Cotnpt. rend, de l'Acad. des Sci., 
1889. Coplin, Phila. Med. Jour., 1899, p. 957. 

2 Edlefsen, Deutsch. Arcb. f. klin. Med., vol. vii. p. 67. Senator, Vircbow's Arcbiv, 
vol. lx. p. 476. Petri. Diss., Berlin, 1876. 

3 B. Bramwell and N. Paton, Laboratory Keports of the Koyal College of Physicians, 
Edinburgh, 1892, vol. iv. p. 47. 



486 THE URINE. 

Albumoses. — Albumoses have frequently been encountered in 
the urine, but are probably more frequently overlooked, as the bodies 
in question are not precipitated on boiling. In former years they 
were commonly regarded as peptones. At present, however, it 
appears to be a well-established fact that true peptones, in the sense 
of Kuhne, viz., true albumins which are not precipitated by salting 
with ammonium sulphate, do not occur in the urine, and the term 
peptonuria should accordingly be abandoned. 

Albumosuria is observed under a great variety of conditions. 
It is thus noted in association with large accumulations of pus 
within the body, and there can be little doubt that the albumo- 
suria is in such instances referable to a disintegration of the pus- 
corpuscles and a resorption of the resulting albumoses. This form 
has hence been termed pyogenic albumosuria. It is principally 
observed during the stage of resolution in cases of croupous pneu- 
monia ; in association with pyothorax, and in cases of epidemic 
cerebrospinal meningitis, as contrasted with the tubercular form. A 
hepatogenic form is noted in connection with diseases of the liver, 
notably acute yellow atrophy. Of its origin, however, nothing is 
known. Formerly, when the condition was looked upon as a pepto- 
nuria, and when it was thought that peptones were retransformed into 
native albumins in the liver, the " peptonuria " was explained upon 
the assumption that the liver had lost its power, and that the " pep- 
tones " accumulated in the blood, and were consequently eliminated 
in the urine. Later researches showed that the transformation of 
peptones into albumins takes place in the intestinal mucous 
membrane, and that the liver probably has no part in the process 
whatsoever. The explanation given had therefore to be aban- 
doned, and, as I have just indicated, we know nothing whatever 
of the origin of this hepatic albumosuria. Possibly it is of an 
enzymatic nature. 

An enterogenic form of albumosuria has been noted in various 
diseases of the intestinal tract, such as typhoid fever, tubercular 
ulceration, carcinoma, etc.; and it is possible that in these cases the 
albumoses are either directly absorbed from disintegrating pus, or 
that the intestine perhaps has in part lost the power of preventing 
the resorption of albumoses as such into the blood. 

A histogenic or hematogenic origin has been ascribed to the albu- 
mosuria which is seen in cases of scurvy, in dermatitis, in various 
forms of poisoning, during the puerperal period and pregnancy, par- 
ticularly following death of the foetus, in various psychoses, in cases 
of carcinomatosis, acute yellow atrophy, etc. 

A renal or vesical form of albumosuria is further noted in which 
the albumoses are derived from contained albumins, owing either to 
the presence of the common proteolytic ferments of the urine or to 
bacterial action, as in decomposing albuminous urines. 



ALBUMINS. 487 

Aside from the conditions already mentioned, albumosuria has 
been observed in various infectious diseases, such as septicaemia, 
pyaemia, diphtheria, measles, scarlatina, acute articular rheumatism, 
mumps, malaria, phthisis ; further, in association with leukaemia, 
nephritis, puerperal parametritis, endocarditis, caries, pleurisy, heart 
disease, apoplexy, myxoedema, carcinomatous peritonitis, in pneumonia 
at the height of the disease and before resolution has set in, in liver 
abscess, etc. 

In the differential diagnosis of suppurative meningitis a positive 
peptone-reaction in the older sense of the word, according to Senator, 
speaks strongly in favor of the existence of this disease. In sup- 
port of this view he cites the case of a young man, the subject of a 
median otitis of long standing, in which symptoms pointing to a 
meningitis — viz., fever, headache, and pains in the neck — were 
present, but in which no " peptonuria " was found to exist, and in 
which an operation revealed the presence of a cholesteatoma. 

A digestive form of albumosuria has recently been described, in 
which albumoses appear in the urine after their ingestion in large 
quantities, and it is claimed that this is observed only in cases of 
ulcerative disease of the intestinal tract. Only a positive result, 
however, is of value. 

Very frequently albumosuria accompanies albuminuria, a condi- 
tion which Senator has termed mixed albuminuria, and it is interest- 
ing to note that the albumosuria may alternate with the albuminuria, 
and may precede or follow the latter. In any case in which albu- 
moses can be demonstrated in the urine the appearance of albumin 
should accordingly be anticipated. 

In all cases of albumosuria the amount of albumose that appears 
in the urine is relatively small, and as a rule cannot be demonstrated 
by the biruret test when applied directly to the native urine. On the 
contrary, it is necessary to isolate the substance more or less definitely 
before deductions can be drawn as to its presence or absence. 

Literature.— Hofineister, Prag. med. Woch., 1889, vol. v. pp. 321 and 325. 
v. Noorden, Lehrbuch d. Path. d. Stoffwechsels, Hirschwald, Berlin, 1893, p. 215. 
Senator, Deutsch. med. Woch., 1895, vol. xxi. p. 217. Stadelmann, Untersuchungen 
iiber Peptonurie, Bergmann, Wiesbaden, 1894. v. Jaksch. Prag. med. Woch., vol. v. 
pp. 292 and 303, and vol. vi. pp. 61, 74, 86, 133, 143; Zeit. f. klin. Med., 1883, vol. vi. 
p. 413. Krehl u. Mattbes, Arch. f. klin. Med., 1895, vol. xlv. p. 54. Maixner, Zeit. f. 
klin. Med., 1884, vol. viii. p. 234. Fisckel, Arch. f. Gynaek., 1884, vol. xxiv. p. 27. 
v. Jaksch, Prag. med. W T och., 1895, vol. xx. p. 430. Katz, Wien. med. Blatter, 1890, 
vol. xiv. L. v. Aldor, Berlin, klin. Woch., 1899, pp. 765 and 785. 

Bence Jones' Albumin. — In association with the occurrence of 
multiple myeloma of the bones, notably when affecting the thora- 
cic skeleton, a peculiar albuminous body is found in the urine, 
which is apparently pathognomonic of the disease in question. It 
was first observed by Bence Jones, and has heretofore been regarded 
as an albumose. From the researches of Magnus Levy and my 



488 THE URINE. 

own investigations, however, it appears that the substance is in 
reality a true albumin, as it yields a proto-albumose on peptic diges- 
tion ; but it differs from all known albumins in its relative solu- 
bility on boiling, and in the readiness with which it dissolves in 
dilute ammonia after precipitation with alcohol. Like casein, it 
contains no hetero-group, but is distinguished from it by the pres- 
ence of a carbohydrate radicle and the probable absence of phos- 
phorus. It is crystallizable, and may occur in the urinary sediment 
in the form of typical spheroliths. 

The amount of the substance which may be found in the urine is 
variable. Some observers have noted an elimination of from 0.25 
to 6.0 pro mille, while others report much larger quantities. In 
Bence Jones' case the elimination rose on one occasion to 6.7 per 
cent., corresponding to a total output of 70 grammes in the twenty- 
four hours — i. e., to nearly as much as the entire amount of the 
albumins of the blood-plasma. 

As regards the origin of the albumin, nothing definite is known, 
but there is reason to suppose that it is not derived from the myel- 
omatous tissue as such. We may imagine, however, that through 
the agency of the cells of the abnormal tissue, viz., their products 
of metabolism, the normal transformation of the ingested albumins 
into tissue-albumins is impeded, resulting in the production of the 
substance in question, which is then eliminated as foreign matter. 

The disease seems to be comparatively rare, and thus far only 
about twenty-eight cases have been reported in which due attention 
was paid to the condition of the urine. Besides these there are 
a few additional cases in which no special note was made of this 
point, though Zahn states that in his case " sometimes more and 
sometimes less albumin" was found. Runeberg also reports that 
the urine of his patient contained much albumin, while the kidneys 
were found practically normal at autopsy. 

As the diagnosis of the disease, in its early stages at least, is 
altogether dependent upon the demonstration of the albumin in 
question, a special examination should be made in this direction in 
all cases of obscure bone-pain, as also in obscure cases of anaemia, 
since Ellinger has shown that at times the disease may take its 
course without the occurrence of local symptoms, while a marked 
anaemia may exist. 

Of special interest in this connection is the fact that Ziilzer claims 
to have succeeded in bringing about the appearance of Bence Jones' 
albumin in the urine of animals by feeding with pyrodin, which is 
known to be a distinct hemolytic poison. 

Literature. — Bence Jones, Med. and Chir. Trans. , 1850, vol. xxxiii. ; and Phil. 
Trans. Royal Soc. of London, 1848. Kiihne, " Ueber Hemialbumose im Harn," Zeit. 
f. Biol., vol. xxix. p. 209. Ellinger, "Ueber d. Vorkommen d. Bence Jones' schen 
Korper im Harn," Arch. f. klin. Med., 1898, vol. lxii. p. 255. Magnus Levy, Zeit. f. 
physiol. Chem., 1900, vol. xxx. p. 200. Hamburger, Johns Hopkins Hosp. Bull., Feb., 

1901. Ziilzer, Berlin, klin. Woch., 1900, p. 894. C. E. Simon, Am. Jour. Med. Sci., 

1902, vol. cxxiii. p. 954. 



ALBUMINS. 489 

pjljtonuria. — To judge from recent investigations of Ito, 1 true 
peptone in the sense of Kiihne, may, after all, occur in the urine 
under pathological conditions. He obtained positive results in 
pneumonia, in advanced cases of phthisis, in ulcer of the stomach, 
and in several women after childbirth. The reaction was most 
intense in the pneumonia cases ; it appeared already before resolu- 
tion occurred, and disappeared a few days after the crisis. In the 
parturient women no reaction was obtained if the examination was 
delayed until after the tenth day. It is noteworthy that in the 
cases examined by Ito the peptonuria was always associated with the 
presence of albumoses (deutero-albumoses), and that the peptone was 
present in still smaller amount than the albumoses. 

Haemoglobin (Methsemoglobin). — Under normal conditions the 
disintegration of the red blood-corpuscles which is constantly taking 
place in the body never results in such a degree of hsemoglobinsemia 
as to be followed by an elimination of haemoglobin in the urine. 
Whenever the destruction of red corpuscles is so extensive, how- 
ever, that the liver is unable to transform into bilirubin all the 
blood-coloring matter set free, hwmoglobinuria occurs. While these 
factors, then — i. e., an excessive destruction of the red blood-cor- 
puscles and an insufficiency on the part of the liver — must be 
regarded as explaining every case of hsemoglobinuria, our knowledge 
of the ultimate causes of such excessive disintegration, as well as 
the manner in which these operate, is limited. Formerly the term 
hcematinuria was applied to this condition. It was shown, however, 
that the pigment eliminated is in reality not haematin, but usually 
methaemoglobin, and only at times haemoglobin, so that the term 
hsemoglobinuria is also ill chosen. 

Most common is the haemoglobinuria produced by certain poisons, 
such as potassium chlorate, arsenious hydride, hydrogen sulphide, 
pyrogallic acid, naphtol, hydrochloric acid, tincture of iodine, carbolic 
acid, carbon monoxide, etc., and also by morels (Helvella esculenta). 

Quite familiar is the hemoglobinuria observed following trans- 
fusion of the blood of animals into man, such as that of the calf 
and lamb ; also the form seen in extensive burns and in insolation. 

While haemoglobinuria may occur in the course of any one of the 
specific infectious diseases, such as scarlatina, icterus gravis, variola 
hemorrhagica, typhoid fever, yellow fever, etc., it is said to be espe- 
cially frequent in cases of malarial intoxication. This view is not 
accepted by many ; Osier, among others, believes that it has fre- 
quently been confounded with malarial hematuria. I have never 
seen an instance of malarial hemoglobinuria, and believe that 
in our more temperate zones it scarcely ever occurs. Bastianello 
asserts that it is likewise rare in Italy, but more common in Sicily 

1 M. Ito, " Ueber d. Vorkommen v. echtem Pepton im Harn," Deutsch. Arch. f. 
klin. Med., 1901, vol. lxxi. p. 29. 



490 THE URINE. 

and Greece, and very common in the tropics. According to the 
same observer, hsemoglobinuria occurs only in infections with the 
sestivo-autumnal parasite. A hemoglobinuria due to quinin is like- 
wise said to exist, but is certainly rare, excepting in patients who 
are suffering or have recently suffered from malarial fever. I have 
seen but one instance of hsemoglobinuria following the ingestion of 
quinin. To judge from the literature upon the subject, there can be 
no doubt that syphilis may under certain conditions be a potent fac- 
tor in the production of hsemoglobinuria. This appears to be par- 
ticularly true of those cases of so-called paroxysmal hsemoglobinuria 
in which bloody urine is voided from time to time, the attacks being 
frequently preceded by chills and fever, so as closely to simulate 
malarial fever. Other factors, also, notably cold, appear to be con- 
cerned in the production of this form. 

The occasional occurrence of hsemoglobinuria in cases of Ray- 
naud's disease, coincident with attacks of an epileptiform character, 
has been referred to in the chapter on the Blood (see page 36). 

Hsemoglobinuria has been observed in a case of leukaemia com- 
plicated by icterus. 

Finally, an epidemic hsemoglobinuria has been described as occur- 
ring in the newborn associated with jaundice, cyanosis, and nervous 
symptoms ; of its catisation we are in ignorance. 

While hsemoglobinuria is rather uncommon, hcematuria is fre- 
quently observed, and w T ill be considered later on, as its recognition 
is not dependent upon the demonstration of the albuminous body, 
" haemoglobin," alone in the urine, but upon the presence of red 
corpuscles, which in hsemoglobinuria are either absent or present 
in only very small numbers. 

Literature. — Hsemoglobinuria : Rosenbach, Berlin, klin. Woch., 1880, vol. xvii. 
pp. 132 and 151. Ehrlich, Zeit. f. klin. Med., 1881, vol. iii. p. 383. Boas, Arch. f. klin. 
Med., 1885, vol. xxxii. p. 355. Kobler u. Obermayer, Zeit. f. klin. Med., 1888, vol. 
xiii. p. 163. 

Fibrin. — The occurrence of fibrin in the urine presupposes the 
presence of fibrinogen and a fibrinogen ic ferment. It is seldom 
seen. According to Neubauer and Vogel, the fibrin may occur 
either as coagulated fibrin or in solution. In the former con- 
dition it is at times observed in the form of blood-coagula, when 
its significance is essentially the same as that of hsematuria in 
general, although it must be remembered that the usual form of 
hsematuria is not associated with the presence of coagula. Colorless 
coagula of fibrin are seen in cases of chyluria or diphtheritic inflam- 
mation of the urinary passages. On the other hand, urines con- 
taining fibrinogenic material in solution are likewise seen but rarely, 
and are characterized by the fact that fibrinous coagula separate out 
only on standing, when they usually cover the bottom of the vessel ; 



ALBUMINS. 491 

but at times they may change the entire bulk of urine into a gelat- 
inous mass. This condition likewise is essentially obseryed in 
cases of chyluria, but may possibly also occur in association with 
nephritis. Lostorfer 1 has reported an instance of this kind, in 
which fibrinous coagulation took place in the clear urine, which con- 
tained much albumin, but no blood. Post-mortem chronic inflam- 
matory changes and amyloidosis of the kidneys was found, while 
the urinary passages proper were intact. 

Nucleo-albumin. — The question "whether or not nucleo-albumin 
is a normal constituent of the urine is still under dispute. Per- 
sonal investigations haye led me to the conclusion that with com- 
plicated methods and large amounts of urine — from 5 to 25 liters — 
it is always possible to demonstrate its presence both under physio- 
logical and pathological conditions. With the usual tests and 
smaller amounts of urine, however, negative results only are obtained 
in strictly normal individuals. According to my experience, tri- 
chloracetic acid, with which Stewart 2 claims to have obtained posi- 
tive results in every one of the one hundred and fifty normal urines 
which he examined, does not precipitate nucleo-albumin when this 
is present in normal amounts. A nucleo-albuminuria recognizable by 
the available tests does not exist under normal conditions. Even under 
pathological conditions nucleo-albumin is by no means always found. 
Sarzin 3 thus was unable to demonstrate its presence in two hundred 
cases which he examined in Senator's clinic. Citron 4 arrived at 
similar results, and of several thousand urines which I have exam- 
ined in this direction positive results were obtained in only a small 
percentage of cases. It is essentially met with in diseases which 
directly or indirectly involve the integrity of the epithelial lining 
of the uriniferous tubules or of the bladder. It has thus been 
frequently found in cases of acute nephritis and associated with 
febrile albuminuria, although its presence even then is not constant. 
In chronic nephritis it is more frequently absent than present. In 
cases of renal hyperemia and cystitis the results are variable. In 
thirty-two icteric urines Obermayer 5 obtained positive results with- 
out exception, and it appears that in leukaemia nucleo-albumin is 
also quite constantly present. During the administration of pyro- 
gallol, naphtol, corrosive sublimate, tar preparations, arsenic, etc., as 
well as in cases of poisoning with anilin and illuminating-gas, large 
amounts of the substance may be found. 

According to my experience, nucleo-albumin is frequently ob- 
tained in cases of so-called functional albuminuria, and it is not 
uncommon to find that this is still present when serum-albumin 
and serum-globulin can no longer be demonstrated, even with the 

1 E. Lostorfer, Wien. klin. Wocb., 1903, Xo. 7. 

2 D. D. Stewart, Med. News, 1894. 

3 D. Sarzin, Leber Nucleo-albuininausscbeidung, Diss., Berlin, 1894. 

4 Ueber Mucin im Ham, Diss., Berlin, 1886. 

5 Obermayer, Centralbl. f. klin. Med., 1892, vol. xiii. p. 1. 



492 THE URINE. 

trichloracetic acid test. Nucleo-albuminuria may thus exist inde- 
pendently of the presence of the more common forms of albumin. 
This observation has also been made by Strauss, who found nucleo- 
albumin only in several cases of cystitis, in one case of chronic in- 
terstitial nephritis, and in one case of emphysema pulmonum with 
renal hyperemia. 

The existence of a hematogenic form of nucleo-albuminuria has 
thus far not been satisfactorily demonstrated. It has been assumed 
that its presence indicates increased epithelial desquamation in some 
portion of the urinary tract — in other words, that it is of cellular 
origin. Matsumoto, however, has shown that even though a urine 
containing numerous epithelial casts, renal epithelial cells, and leu- 
cocytes be allowed to stand for some time, a substance which can be 
precipitated with acetic acid either does not occur at all or only in very 
small quantity. He has rendered it very probable that the substance 
which can be precipitated from pathological urines by means of acetic 
acid is largely fibrinogen and euglobulin. He adds that nucleo-albumin 
may be present simultaneously, but in comparison to the other two 
substances it is of secondary importance and is rarely seen. 

Histon and Nucleohiston. — Kolisch and Burian l were able to 
demonstrate the presence of histon in a case of leukaemia in which 
it was constantly present. More recently Krehl and Matthes 2 claim 
to have isolated the same substance in various febrile diseases, such 
as acute peritonitis, following appendicitis, in croupous pneumonia, 
erysipelas, and scarlatina. It is an albuminous body, and was first 
discovered by Kossel in the red blood-corpuscles of the goose. It 
exists in the leucocytes of human blood in combination with the acid 
leukonuclein, constituting the so-called nucleohiston of Lilienfeld. 

It is not clear in what manner the histonuria is produced ; so 
much, however, seems certain, that it is not solely dependent upon 
increased destruction of leucocytes. 

Nucleohiston itself has been found in the urine in a case of 
pseudoleukemia, by Jolles. 3 

Tests for Albumin. — The recognition of the various albuminous 
bodies which may occur in the urine is based partly upon their 
direct precipitation and partly upon color-reactions when treated 
with certain reagents. 

The number of tests which have from time to time been sug- 
gested is large ; many of them after a brief period of use have been 
discarded as useless or uncertain, while others have been employed 
only occasionally, and have not received the recognition which they 

1 R. Kolisch u. E. Burian, " Ueber d. Eiweisskorper d. leukanrischen Harns," etc., 
Zeit= f. klin. Med., vol. xxix. p. 374. 

2 L. Krehl u. M. Matthes, ''Ueber febrile Albumosurie," Deutsch. Arch. f. klin. 
Med., vol. liv. p. 508. 

3 A. Jolles, Ber. d. deutsch. chem. Gesellsch., vol. xxx. p. 172; Zeit. f. klin. Med., 
vol. xxxiv. p. 53. 



ALBUMINS. 



493 



deserve, from the fact that simpler tests exist, that they do not 
possess sufficient delicacy, or that in some instances it is too great. 
In the following pages no attempt is made to describe all of 
these tests, and attention will be directed only to those which are 
generally used, and which clinical experience has proved to be of 
value, precedence being given to those which have been longest in 
While some of these are applicable for demonstrating the 



use. 



Fig. 11]. 



presence of more than one form of albumin, special tests will also 
be described whereby the various albumins may be individually 
recognized. 

In every case the urine should be carefully filtered, so as to free 
it from any morphological elements, etc., present. To this end, it 
is generally sufficient to pass the urine through one or two layers 
of Swedish filter-paper. Frequently, however, a clear specimen 
cannot be obtained in this manner ; it is then advisable to shake the 
urine with burnt magnesia or talcum, or to mix it with scraps of 
filter-paper, when it is filtered as usual. 

Tests for Serum-albumin. — The Nitric Acid Test 1 (Fig. 111). 
— The value of this test, properly ap- 
plied, cannot be overestimated, as 
it is not only simple, but yields an 
amount of information that can 
otherwise be gained only with dif- 
ficulty. Usually the student is ad- 
vised to make use of a test-tube par- 
tially filled with urine, along the 
sides of which concentrated, chemi- 
cally pure nitric acid is allowed to 
flow, so as to form a layer at the 
bottom of the tube, when in the 
presence of serum-albumin a distinct 
white ring appears at the zone of con- 
tact between the two liquids (Heller's 
test). The pictures thus obtained can- 
not be compared, however, with those 
seen when the apparently trivial change 
is made of using a conical glass of 
about 2 ounces capacity instead of the 
test-tube. About 20 c.c. of urine are 
placed in the glass, when 6 to 10 c.c. 
of nitric acid are added by means of a 

pipette, which is carried to the bottom of the vessel ; the acid is slowly 
allowed to escape by diminishing the pressure of the finger upon the 
tube. When this is carefully done the nitric acid forms a. distinct 
zone beneath the urine. In the presence of albumin the white 

1 J. F. Heller, Arch. f. physiol. u. path. Chem. u. Micros., 1852, vol. v. p. 169. A. 
Robin, Urologie clinique de la fievre typhoide, Paris, 1877. 




Nitric acid test. 



494 THE URINE. 

ring then appears, and varies in extent and intensity with the 
amount of albumin present (Plate XVIIL, Fig. 1). If now the 
contents of the glass are allowed to stand undisturbed — and if small 
amounts are present, these appear only oh standing for several 
minutes — it will be observed that the cloudiness gradually extends 
upward ; and if much albumin is present, it may be seen to rise 
into the supernatant liquid in the form of small, irregular col- 
umns. This appearance is possibly referable to the partial decom- 
position of uric acid by means of nitric acid, nitrogen and carbon 
dioxide being set free, which, rising to the surface in the form of 
small bubbles, carry the nitric acid upward ; coming into contact 
with albumin in solution, this is then precipitated. 

An excess of uric acid is indicated by the appearance, within five 
to ten minutes after addition of the nitric acid, of a distinct ring in 
the clear urine, about 1 to 2 cm. above the zone of contact, which is 
similar in appearance to that due to albumin. If this ring (Plate 
XVIII., Figs. 1, 2, and 3), which has been appropriately compared 
to a communion wafer, does not appear within five to ten minutes, it 
may be assumed that uric acid is present in diminished amount. 
The degree of increase, on the other hand, may be determined by 
the size of the ring, it being presupposed that the same quantities 
of urine and of the reagent are employed in every case. 

Should more than 25 grammes of urea be contained in a liter of 
the urine examined, an appearance like hoarfrost will be noted on 
the sides of the vessel, which is due to the formation of urea nitrate. 
Spangles of the same substance appear only in the presence of at 
least 45 grammes ; and if 50 grammes or more of urea are contained 
in the liter, a dense mass of urea nitrate may be seen to separate out. 

Biliary urine, when treated with nitric acid containing a little 
nitrous acid, shows the color-play referable to the action of nitric 
acid upon bilirubin (Plate XVIII., Fig. 4). The production of the 
colors (red, yellow, green, blue, and violet) takes place from above 
downward, the green color being the most characteristic ; in the 
absence of the latter the presence of biliary pigment may be posi- 
tively excluded. The presence of albumin is not objectionable, as 
the color-play takes place beneath the albuminous disk. 

In normal urine a transparent ring is also obtained, presenting a 
peach-blossom-red ; the intensity of this may vary, however, from a 
faint rose to a pronounced brick color, and is referable to normal 
urinary pigment (Plate XVIII., Fig. 5). In the presence of uro- 
bilin, on the other hand, this ring presents a distinct mahogany color. 

Indican is indicated by the appearance of a violet ring (Plate 
XVIIL, Fig. 2) situated above that referable to the normal urinary 
pigment. Its intensity varies with the amount present, from a light 
blue to a deep indigo. 

The milky cloud at the zone of contact of the two fluids may be 
referable not only to the presence of serum -albumin, but also of 



PLATE XVIII 



FIG. 2. 



FIG. 4. 



FIG. 1. 





FIG. 3. 



PCTPPPf 



FIG. 8. 





ALBUMINS. 495 

globulin and albuinoses (propeptones), while a negative reaction will 
generally indicate the absence of these bodies. That the uric acid 
ring will be mistaken for albumin is hardly likely if it is remem- 
bered that this never first appears at the zone of contact of the two 
fluids, but always in the uppermost portion of the urine. It is true 
that urines are occasionally observed in which the separation of uric 
acid takes place so suddenly that within a minute or two the entire 
urinous portion of the mixture is completely clouded, resembling the 
appearance presented by a highly albuminous urine. Such an exces- 
sive elimination of uric acid is uncommon, however, and it is to be 
remembered that with uric acid the cloudiness extends from above 
downward, and never from below upward, as is the case with albu- 
min. Should any doubt be felt, it is only necessary to remove a 
few cubic centimeters of this cloudy urine by means of a pipette and 
to heat it gently in a test-tube, when the urine will clear up entirely 
if the precipitate is due to uric acid, while if caused by albumin it 
will remain or become more intense. Should the precipitate caused 
by nitric acid consist of album oses, it will also clear up entirely, 
to reappear on cooling, the fluid at the same time assuming a 
markedly yellow color. The occurrence of a distinctly yellow color 
in the urine, moreover, which is only partially cleared upon the 
application of heat (and be it remembered that a much higher tem- 
perature is necessary for the solution of a precipitate referable to 
albumoses than of one due to urates), will indicate the existence of 
a mixed albuminuria — i. e., the presence of coagulable albumin and 
albumoses. 

Nitric acid may also cause a precipitation of certain resinous bodies, 
such as those contained in turpentine, balsam of copaiba aud tolu, etc. 
If any doubt is felt, the mixture should be shaken with alcohol, 
when the precipitate caused by these substances is at once dissolved. 
The mucinous body — nucleo-albumin — which is at times found in 



DESCEIPTION OF PLATE XVIII. 
The Nitric Acid Test as Applied to the Urine. 

Fig. 1. — The light, colorless ring in the clear urine above shows a slight increase 
in the amount of uric acid ; the large white band denotes a large amount of albumin, 
bordering upon a colored ring, referable partly to indican (blue) and partly to uro- 
rosein. 

Fig. 2. — The light ring in the clear urine above denotes a slight increase in the 
amount of uric acid. The bluish-black band is referable to an enormous increase 
in the amount of indican. (Ileus.) 

Fig. 3. — The broad, light band in the clear urine above is referable to an enor- 
mous increase in the amount of uric acid. (Laparotomy.) 

Fig. 4. — The color-play referable to the presence of bilirubin is shown in a dia- 
grammatic manner. 

Fig. 5. — The colored ring is referable to the presence of normal urinary coloring- 
matter. 



496 THE URINE. 

the urine is also precipitated by nitric acid, but need not occupy our 
attention at this place. From what has been said, it is manifest 
that the employment of the nitric acid test in the manner indicated 
furnishes much valuable information, and the adoption of the method 
as described not only by hospital students, but by general practi- 
tioners as well, cannot be too strongly urged. 

Boiling Test. — A few cubic centimeters of urine are boiled in a 
test-tube and then treated with a few drops of concentrated nitric acid, 
no matter whether a precipitate has occurred upon boiling or not. 
If albumin is present, this will separate out as a flaky precipitate, 
which consists of serum-albumin frequently mixed with serum- 
globulin. It is true that albuminous urines will generally yield a 
precipitate on boiling alone ; but it must be remembered that unless 
the reaction is decidedly acid a precipitation of normal calcium 
phosphate may occur, owing to the fact that the reaction of the urine 
upon boiling becomes less acid from escape of the carbonic acid held 
in solution. In urines presenting an alkaline or amphoteric reac- 
tion this is very frequently noted, and might give rise to confusion, 
as the precipitate due to calcium phosphate closely resembles that 
referable to albumin. Care must hence be taken to insure a dis- 
tinctly acid reaction, which is best accomplished by the addition of 
nitric acid, when a precipitate referable to phosphates is at once dis- 
solved, while one due to albumin remains, and may even become 
more marked. The quantity to be added should usually be equiva- 
lent to about 0.05 to 0.1 of the volume of urine. Under no con- 
dition should the acid be added before boiling, nor should the 
urine be boiled after its addition, as small amounts of albumin will 
otherwise be overlooked, owing to the fact that hot nitric acid dis- 
solves the precipitate to a certain degree. If, after addition of 
the nitric acid the urine turns a distinct yellow, and if then upon 
cooling a white precipitate appears, the presence of albumoses may 
be inferred. Uric acid will probably never cause confusion, as this 
separates out only upon cooling, and then presents a dark-brown 
color. As in the case of the nitric acid test, so also here, a pre- 
cipitation of certain resins is noted at times which may be recognized 
by their solubility in alcohol. Albumoses are also precipitated upon 
the application of heat, but such precipitates again dissolve when 
the temperature approaches the boiling-point (see page 502). 

Should acetic acid be used instead of nitric acid, great care must 
be taken to avoid an excess, as otherwise the albumin will be dis- 
solved. As this danger diminishes the greater the quantity of salts 
contained in the urine, it is advisable to treat the urine first with a 
few drops of acetic acid until a distinctly acid reaction is obtained, 
and then to add one-sixth its volume of a saturated solution of 
sodium chloride, magnesium sulphate, or sodium sulphate, when 
upon boiling a precipitation of the albumin will occur. Carried 



ALBUMINS. 497 

out in this manner, the test is absolutely certain and will dem- 
onstrate even minimal amounts of albumin. If an equal volume 
of a saturated solution of common salt is added to the acidified urine, 
albumoses are also precipitated, but the precipitate dissolves on 
boiling. 

The Potassium Ferrocyanide Test. — A few cubic centi- 
meters of urine are strongly acidified with acetic acid (sp. gr. 1.064) 
and treated with a few drops of a 10 per ceut. solution of potassium 
ferrocyanide, when, in the presence of but little albumin, a faint 
turbidity, or, if much albumin is present, a flaky precipitate, is 
noted, which is best recognized by comparison with a tube contain- 
ing some of the pure filtered urine, both tubes being held against 
a black background. Concentrated urines should be previously 
diluted with water, as albumoses, like serum-albumin and serum- 
globulin, which may be precipitated in this manner, otherwise re- 
main in solution. Here, also, as in the tests described, the presence 
of albumoses may be inferred if the precipitate disappears upon 
boiling, while a partial clearing up, on the other hand, indicates the 
presence of albumoses and coagulable albumin. 

At times the addition of acetic acid by itself is followed by the 
appearance of a cloud in the urine, which may be due to urates or 
to urinary mucin (nucleo-albumin), as already mentioned. In such 
cases the urine should be refiltered, diluted with water, and the test 
again applied. 

v. Jaksch advises the careful addition, by means of a pipette, of 
a few cubic centimeters of fairly concentrated acetic acid, to which a 
little potassium ferrocyanide has been added, when the albumin, as in 
Heller's test, is seen to form a ring at the zone of contact between 
the two fluids. Instead of potassium ferrocyanide, potassium plat- 
inocyanide may also be employed, and has the advantage that the 
test-solution is colorless. 

The Trichloracetic Acid Test. 1 — This test is undoubtedly 
the most delicate of those so far described, but not so delicate that 
a trace of albumin or nucleo-albumin can be demonstrated in every 
urine. An experience based upon the examination of several thou- 
sand urines with this reagent warrants my speaking with a certain 
degree of confidence upon the subject. Very frequently it is pos- 
sible with this method to demonstrate albumin in urines in which 
the more common tests yield negative results, but in which tube- 
casts may nevertheless be found upon microscopical examination. 
The test is applied as follows : by means of a pipette 1 or 2 c.c. of 
an aqueous solution of the reagent (sp. gr. 1.147) are carried to the 
bottom of a test-tube containing the carefully filtered urine, so as to 
form a layer beneath the urine. In the presence of albumin a white 

1 F. Obermayer, Wien. med. Jahrbiich, 1888, p. 375. D. M. Eeese, Johns Hopkins 
Hosp. Bull., 1890. 

32 



498 THE URINE. 

ring will be seen to form at the zone of contact between the two fluids, 
varying in intensity with the amount of albumin present. So far as 
the test for albumin is concerned, this reagent possesses an advantage 
over nitric acid in that the colored rings, which are so confusing 
to the inexperienced, are commonly not observed. Serum-albumin, 
serum-globulin, and albumoses are precipitated, the presence of the 
latter being recognized, as in the previous tests, by the fact that the 
precipitate disappears upon boiling and reappears on cooling. A 
cloud, referable to uric acid, also appears if this is present in exces- 
sive amounts, but disappears upon the application of gentle heat. 
A previous dilution of the urine, moreover, guards against its occur- 
rence. 

Other tests have also been suggested for the detection of albumin 
in the urine, such as the metaphosphoric acid test, the phenol, tannic 
acid, and picric acid tests, that with Tanret's reagent, phospho- 
tungstic and phosphomolybdic acids, and quite recently Spiegler's 
reagent. 

Of these, only the picric acid and Spiegler's test will be con- 
sidered. 

Picric Acid Test. — The picric acid test is not applicable as a 
test for albumin as such, and is mentioned in this connection only 
because the same reagent is employed with Esbach's quantitative 
method. This is composed of 10 grammes of picric acid and 20 
grammes of crystallized citric acid, dissolved in a, liter of distilled 
water. If to this solution albuminous urine is added, the mixture 
is rendered turbid, and after some time a sediment which consists 
not only of albumins, but also of uric acid, kreatinin, and other 
extractives, will form at the bottom of the tube (see Quantitative 
Estimation of Albumin). 

Spiegler's Test. 1 — Spiegler's reagent consists of 8 parts by 
weight of mercuric chloride, 4 parts of tartaric acid, and 200 parts 
of water, in which 20 parts of cane-sugar are further dissolved, so 
as to increase the specific gravity of the reagent and permit of its 
being employed, like Heller's test, even in concentrated urines. 
One-third of a test-tube is filled with the reagent, and the urine 
carefully placed above this by allowing it to flow slowly down the 
side of the tube ; in the presence of albumin a sharply defined white 
ring will be observed where the two liquids are in contact. Peptone 
gives no reaction, while albumoses are precipitated and may be 
recognized as indicated above. Unfortunately the reagent will also 
precipitate nucleo-albumin. 

Special Test for Serum-albumin. — Should it be desired, for 
any reason, to demonstrate serum-albumin alone, the urine is ren- 
dered amphoteric or faintly alkaline with sodium hydrate, and is then 
saturated with magnesium sulphate in substance, in order to remove 
any globulin. The filtrate is strongly acidified with acetic acid, 
i Spiegler, Wien. klin. Wocb., 1892, vol. v. p. 26. 



ALBUMINS. 499 

when a flaky precipitate, appearing upon boiling, will indicate the 
presence of serum-albumin. 

Patein's albumin differs from the common serum-albumin in being 
soluble in acetic acid. 1 

Very often, as in the examination for sugar, it is necessary to 
remove any coagulable albumin that may be present, to which end 
the urine is rendered distinctly acid with acetic acid and boiled. An 
examination of the nitrate with potassium ferrocyanide, if the 
amount of acetic acid added was just sufficient, will then yield a 
negative result (see page 497). 

Quantitative Estimation of Albumin. — For the quantitative esti- 
mation of albumin a large number of methods have been devised, 
which fact in itself is sufficient to indicate that the majority of them, 
at least, are unsatisfactory. 

Old Method by Boiling. — If comparative results only are de- 
sired, a definite amount of urine is boiled after acidifying with acetic 
acid ; the albumin is allowed to settle for twenty-four hours. For 
this purpose Neubauer suggests the use of glass tubes measuring one- 
half to three-quarters of an inch in diameter, which are closed at the 
lower end with a cork. Ordinary test-tubes answer perfectly well, 
but care should be taken that the same quantity of urine is used in 
each case. The tubes are corked and kept for several days for com- 
parison. The results, of course, express only the relative amount 
of albumin present, and it should be remembered that the error 
incurred may amount to as much as 30 or even 50 per cent, of 
the quantity that is found by gravimetric analysis. This is owing 
to the fact that sometimes the albumin separates out in large flakes, 
and at other times in small flakes, and that the degree of precipita- 
tion is also influenced by the specific gravity of the supernatant 
urine. 

Volumetric Method of Wassiliew. 2 — This method can be 
recommended for the quantitative estimation of albumin, as it is 
both simple and accurate. 

Ten to 20 c.c. of urine, which are best diluted to 50 c.c. with 
distilled water, are treated with 2 drops of a 1 per cent, aqueous 
solution of true yellow (Echtgelb 3 of Griibler), and then titrated 
with a 12.5 per cent, solution of salicyl-sulphonic acid until a dis- 
tinct brick-red color is obtained. The number of cubic centimeters 
of the reagent employed, multiplied by 0.00006, will indicate the 
amount of albumin in the 10 or 20 c.c. of urine examined. If the 
urine is alkaline, it should first be slightly acidified with acetic acid. 

1 Patein, " Acetosoluble Albumin in the Urine," Compt. rend, de l'Acad. des Sci., 
1889. Coplin, Phila. Med. Jour., 1899, p. 957. 

2 Wassiliew, Eshenedelnik, 1896, No. 26 ; St. Petersburg, med. Woch., 1897, Beilage, 
p. 4. 

3 Echtgelb is a mixture of amidoazobenzol disulphonate and sodium monosul- 
phonate. 



500 



THE URINE. 



Fig. 112. 



K 



fr-q 



Esbach's Method. 1 — The reagent is composed of 10 grammes 
of picric acid and 20 grammes of citric acid, dissolved in 1000 c.c. 
of distilled water. Special tabes, termed albnminimeters 
(Fig. 112), are employed, which bear two marks, one, U, 
indicating the point to which urine must be added, and one, 
R, the point to which the reagent is added. The lower 
portion of the tube up to U bears a scale reading from 
1 to 7, corresponding to the amount of albumin pro mille. 
The tube is filled to U with the filtered albuminous 
urine, and the reagent added until the point R is 
reached. The tube is then closed with a stopper, inverted 
twelve times, and set aside for twenty-four hours. At 
the expiration of this time serum-albumin, serum-globulin, 
and albumoses, as well as uric acid and kreatinin, will 
have settled, when the amount pro mille in grammes may 
be directly read oif from the scale. A few precautions 
must, however, be observed in order to obtain as accurate 
results as possible. The reaction of the urine should be 
acid, and if this is not the case acetic acid is added. Its 
specific gravity should not exceed 1.006 or 1.008, the 
proper density being obtained by diluting with water. 
The amount of albumin in the specimen should not ex- 
ceed 0.4 per cent. ; if more be present, as determined by 
a imeter?" a preliminary test, the urine should be diluted. Most 
important, furthermore, is the temperature of the room. 
This should be 15° C. ; variations from this point are apt to give 
rise to inaccurate results, which, according to Christensen, may 
amount to 100 per cent, in the case of a deviation of only 5 
degrees C. It is thus clear that as generally employed in the clinical 
laboratory the method will only give approximate results. 

The Differential Density Method, 2 — More accurate results 
may be obtained with the following method, which is based upon the 
diminution in the specific gravity of the urine after the removal of 
all albumin, and its comparison with the specific gravity observed 
before. To this end, the urine is treated with a sufficient amount of 
acetic acid to insure complete precipitation of the albumin (see 
below), when its specific gravity is noted. It is then brought to 
the boiling-point, care being taken to guard against evaporation by 
placing the urine in an ordinary medicine-bottle ; this is closed with 
a rubber stopper that has been previously boiled in a solution of 
sodium hydrate and washed free from alkali, the stopper being tightly 
fastened with a cord or wire. Thus prepared, the bottle is kept in 
boiling water for ten to fifteen minutes. The urine is filtered on 
cooling, evaporation being again carefully guarded against by filter- 



1 Guttmann, Berlin, klin. Woch., 1886, vol. xxiii. p. 117. 

2 Huppert u. Zakor, Zeit. f. physiol. Chem.., 1886, vol. xii. pp. 467 and 484. 



ALBUMINS. 501 

ing into a bottle through a funnel which has been passed through a 
closely fitting stopper ; the funnel is kept covered with a plate of 
glass. The specific gravity is then again determined, and it is best 
in both cases to use a pyknometer. (An accurate hydrometer, grad- 
uated to the fourth decimal, may, however, also be used.) The 
decrease in the specific gravity, multiplied by 400, will indicate the 
number of grammes of albumin in 100 c.c. of urine. 

Gravimetric Method. — If accuracy is required, the amount of 
albumin must be determined gravimetrically as follows : a certain 
quantity of urine, after having been acidified with an amount of acetic 
acid sufficient to insure complete precipitation of all albumin, is 
boiled ; the albumin is then filtered off, dried, and weighed. For this 
purpose, 500 to 1000 c.c. of carefully filtered urine should be avail- 
able. A specimen of this, if already acid, is placed in a test-tube, in 
boiling water, until coagulation takes place, when it is further heated 
over the free flame and filtered. The filtrate is then tested with 
acetic acid and potassium ferrocyanide. Should no albumin be 
thus demonstrable, the entire amount of urine is treated in the same 
manner, and requires no further addition of acetic acid. If, how- 
ever, the test yields a positive result, it is apparent that the urine 
was not sufficiently acid. The entire volume is then treated with a 
30 to 50 per cent, solution of acetic acid, drop by drop, the mixture 
being thoroughly stirred and specimens tested from time to time, as 
described. When, finally, the urine remains clear or shows only a 
faint turbidity, 100 c.c. or less, according to the amount of albumin 
present, are first heated in boiliug water until the albumin begins to 
separate out in flakes, and then carefully brought to the boiling-point 
over the free flame. The supernatant urine is decanted through a 
filter, which has been previously dried at 120° to 130° C. and 
accurately weighed, when the whole amount of the precipitate is 
brought upon the filter. Any albumin remaining in the beaker is 
detached from its sides by means of a glass rod tipped with a piece 
of rubber tubing, and collected by the aid of hot water. The entire 
precipitate is now thoroughly washed with hot water until the wash- 
ings no longer become turbid when treated with a drop of nitric acid 
and silver nitrate ; in other words, until the chlorides have been 
completely removed. The precipitate is further washed with alco- 
hol and finally with ether to remove any fats that may be present, 
when it is dried at 120° to 130° C. until a constant weight is 
reached. If still greater accuracy is required, the dried and weighed 
precipitate is incinerated to determine the amount of mineral ash in 
combination with the albumin, which is then deducted from the total 
weight. The most accurate results are obtained if not more than 0.2 to 
0.3 gramme of albumin is contained in the amount of urine employed. 
A smaller quantity than 100 c.c. should hence be used if a previous 
test with Esbach's albuminimeter shows a higher percentage. 



502 THE URINE. 

A glass-wool filter insures a more rapid process of drying — twenty- 
four to thirty hours ; but care must then be had that this is properly 
prepared, so as to guard against a loss of the wool while washing. 

Method by Centrifugation. — This presupposes a constant 
speed , and hence an electrical centrifuge is a prerequisite, which is 
an objection to the general adoption of the method. But even with 
its aid results are not always obtained which are accurate. 

Test for Serum-globulin and its Quantitative Estimation. — To test 
for serum-globulin the urine is rendered alkaline by the addition of 
ammonium hydrate, any phosphates that may thus be thrown down 
being filtered oif on standing. The urine is then treated with an 
equal volume of a saturated solution of ammonium sulphate, when 
the occurrence of a precipitate will indicate the presence of the 
globulin. Ammonium urate may likewise separate out, but this 
occurs later. 

According to Paton, the following test may also be employed : the 
urine after having been rendered alkaline with sodium hydrate, — 
any phosphates which may separate out are filtered off, — is carefully 
poured down the side of a test-tube containing a saturated solu- 
tion of sodium sulphate, so as to form a layer above this, when in 
the presence of serum-globulin a white ring will appear at the zone 
of contact. 

If a quantitative estimation of the globulin is to be made, the pre- 
cipitate thus obtained, after about one hour's standing, is collected 
on a dried and weighed filter, and washed thoroughly with a one- 
half saturated solution of ammonium sulphate until a specimen 
of the washings treated with acetic acid and potassium ferrocy- 
anide no longer gives a precipitate. It is then treated as directed 
in the method employed for the quantitative estimation of serum- 
albumin. 

Tests for Albumoses. — A small amount of urine is strongly acidi- 
fied with acetic acid and treated with an equal volume of a saturated 
solution of common salt. In the presence of albumoses a precipitate 
occurs, which dissolves on boiling and reappears on cooling. If 
serum-albumin also be present, which is usually the case, the hot 
liquid must be filtered. The albumoses are found in the filtrate and 
appear on cooling. If the hot filtrate, moreover, is rendered alkaline 
with a solution of sodium hydrate, a red color develops upon the 
addition of a very dilute solution of cupric sulphate, added drop by 
drop (biuret reaction). On boiling with MiUon's reagent a red color 
is also obtained. This reagent is prepared by dissolving 1 part of 
mercury in 2 parts of nitric acid of a specific gravity of 1.42, and 
diluting with 2 volumes of distilled water. 

Salkowski's Method. — Fifty c.c. of urine are acidified in a 

1 E. Salkowski, " Ueber d. Nactaweis d. Peptons (Albumosen) im Harn u. d. Darstel- 
luug d. Urobilins," Berlin, klin. Woch., 1887, p. 353. 



ALBUMINS. 503 

beaker with 5 c.c. of hydrochloric acid, and precipitated with phos- 
photungstic acid, the mixture being heated over the free flame, when 
in a few minutes the precipitate will form a resinous mass which 
closely adheres to the bottom of the vessel. The supernatant fluid 
is decanted, and the mass at the bottom, which now becomes granular, 
washed twice with distilled water, which is likewise removed by 
decantation. The precipitate is then covered with about 8 ex. of 
distilled water, and treated with 0.5 c.c. of a sodium hydrate solu- 
tion (sp. gr. 1.16). Upon shaking the beaker the mass will dissolve, 
the solution assuming a dark-blue color. This is heated on the free 
flame until the blue color turns to a dirty, grayish-yellow ; the solu- 
tion at the same time becomes turbid, but at times may turn yellow 
and remain clear. This discoloration may be hastened by the further 
addition of a few drops of sodium hydrate solution. As soon as 
this point has been reached, some of the liquid is placed in a test- 
tube, allowed to cool, and then treated with a very dilute solution 
of cupric sulphate (1 to 2 per cent.) drop by drop ; in the presence 
of peptones the solution assumes a bright-red color, which may be 
brought out still more strongly if the specimen is now filtered. If 
albumin or much mucin is present, these bodies must first be re- 
moved (see pages 499 and 506) ; but the quantity of urine employed 
is so small that the mucin can usually be disregarded. With this 
method, which occupies only about five minutes, 0.015 gramme of 
peptones pro 100 c.c. may be demonstrated without difficulty. 

Salkowski has recently pointed out that urines which are very 
rich in urobilin, as in pneumonia, may give rise to the biuret reac- 
tion even when albumoses are absent. The coloring-matter, it is 
true, may be removed entirely by precipitation with lead acetate or 
subacetate, but unfortunately a portion of the albumoses is also 
carried down, and the substance may thus escape detection when 
present only in small amounts. He hence suggests that smaller 
quantities of urine, such as 10 c.c, be employed in the test. The 
reaction is then not so well marked, but the results are more re- 
liable. 

Bang's Method. — This method has recently been introduced, 
and is said to be free from the objections attaching to the one pro- 
posed by Salkowski. Ten c.c. of urine are heated in a test-tube 
with 8 grammes of finely powdered ammonium sulphate until the 
salt has been dissolved ; the fluid is then boiled for a moment. The 
hot fluid is centrifugated for one-half to one minute, the supernatant 
fluid poured off, and the sediment stirred with alcohol in an agate 
mortar. The alcohol is poured off, and the residue dissolved in a 
little water ; the solution is boiled and filtered, and the filtrate tested 
with sodium hydrate solution and cupric sulphate as described. 
Should the urine be especially rich in urobilin — i. e., manifesting 
a well-marked fluorescence with zinc chloride and ammonia — it is 



504 THE URINE. 

best to extract the final aqueous solution with chloroform by shak- 
ing, and to pour off the supernatant fluid, when this is tested with 
cupric sulphate. In this manner it is possible to demonstrate the 
presence of albumoses in a dilution of 1 : 4000-5000. Other con- 
stituents of the urine, with the exception of hsematoporphyrin, do 
not interfere with the test. Should hsematoporphyrin be present, 
however, which may be suspected if a red alcoholic extract is obtained, 
the urine must first be precipitated with barium chloride. The fil- 
trate, which contains the albumoses, is then examined as described. 

If a centrifuge is not available, the urine is boiled with the ammo- 
nium sulphate, when a portion of the albumoses will remain on the 
sides of the tube as a sticky mass. This is washed with alcohol, 
and if necessary with chloroform, dissolved in water, and tested for 
biuret. 

The alcoholic extract may also be used for testing for urobilin. 
To this end, it is only necessary to add a few drops of a solution 
of zinc chloride, when in the presence of urobilin a beautiful fluores- 
cence will be observed. The test is extremely delicate. 1 

Examination for True Peptone. 2 — To demonstrate the presence of 
true peptone in the urine, about 300 c.c. of filtered acid urine are 
saturated on a water-bath with ammonium sulphate at a temperature 
between 60° and 70° C. On cooling, the mixture is filtered, the 
filtrate is alkalinized with a dilute solution of sodium carbonate, 
again saturated between 60° and 70° C. with ammonium sulphate, 
filtered on cooling, the filtrate neutralized with very dilute acetic acid, 
again saturated with the salt between 40° and 50° C, and finally 
again filtered on cooling. The final filtrate is diluted with an equal 
volume of distilled water and treated with a freshly prepared solu- 
tion of tannic acid, which is added drop by drop, care being taken 
to avoid an excess. The precipitate is filtered off the next day, 
dried in the desiccator upon the filter, powdered, and covered in a 
porcelain crucible with a small amount of baryta-water to which a 
little finely powdered baryta is added. The mixture is placed on a 
boiling water-bath for three minutes, and after one or two hours it 
is filtered. If necessary, the solution is decolorized with neutral 
lead acetate. The biuret test is finally applied, and if positive, 
indicates the presence of peptone in the sense of Kiihne. 

Tests for Bence Jones' Albumin. — The presence of Bence Jones' 
albumin is usually discovered on slowly heating the urine to the 
boiling-point. It will then be noted that at a temperature of from 
50° to 60° C. a more or less intense, milky turbidity develops, which 
on subsequent boiling either disappears entirely or partially, and 
reappears on cooling. The degree to which the urine clears on 

1 E. Bang, "Eine neue Methode zum Nachweis d. Albumosen im Harn," Deutseh. 
med. Woch., 1898, p. 17. 

2 Ito, loc. cit. 



ALBUMINS. 505 

boiling differs in different cases. As I have just stated, the turbid- 
ity may disappear entirely ; but, on the other hand, urines are met 
with in which even a partial clearing can scarcely be made out. 
This is apparently dependent upon the degree of acidity of the urine, 
the amount of mineral salts and of urea present, and probably also 
upon other and still unknown factors. 

Upon the addition of a drop of nitric acid to a few cubic centi- 
meters of such urine a temporary turbidity develops, which disap- 
pears on shaking, but persists if a little more of the acid is added. 
If now the mixture is heated, the albumin first coagulates to a dense 
mass ; on boiling, this dissolves, and after a while the liquid becomes 
almost entirely clear, while the turbidity returns, as before, on sub- 
sequent cooling. Similar reactions are obtained with all the common 
reagents for albumin. 

For its complete identification, the albumin should be isolated 
and further examined as follows : larger amounts of urine are pre- 
cipitated by the addition of one and one-half to two volumes of 
96 per cent, alcohol, or by treating with two volumes of a saturated 
solution of ammonium sulphate. In either event the total amount 
of albumin is thrown down. This is then washed with alcohol and 
ether, and dried over sulphuric acid. To purify the substance, it is 
dissolved in boiling water, by the aid of a few drops of a dilute 
solution of sodium carbonate, and dialyzed to running and then to 
distilled water until free from mineral salts. It is then reprecipi- 
tated with alcohol (if necessary, after the addition of a drop or two 
of a dilute solution of hydrochloric acid), washed with absolute 
alcohol and ether, and dried. Thus purified, the albumin is prac- 
tically insoluble in distilled water or saline solution at ordinary tem- 
perature, and only sparingly so at the boiling-point. In boiling 
water, however, it dissolves with comparative ease after the addi- 
tion of a few drops of sodium carbonate solution. On neutraliza- 
tion no precipitate occurs if a sufficient amount of water is present. 
If such a neutral solution is heated, no change occurs ; but if it is 
now acidified and a certain amount of salt added, the typical reaction 
appears on heating, viz., precipitation between 50° and 60° C. (even 
between 40° and 50° C. if a sufficient amount of salt is present), 
clearing on boiling, and reprecipitation on cooling. 

On digestion with pepsin-hydrochloric acid, as I have said, a 
proto-albumose is obtained among the early products of digestion, 
while a hetero-albumose is not formed. 

Test for (Mucin) Nucleo-albumin. — It has been generally supposed 
that the substance which is precipitated on adding strong acetic acid 
to certain pathological urines, when diluted two or three times with 
water, is nucleo-albumin, the precipitate being soluble or largely so 
in an excess of the reagent. Matsumoto, 1 however, has recently 

1 Matsumoto. " Ueber d. durch Essigsaure ausfallbare Eiweissubstauz in patbo- 
logiscben Harnen," Deutscb. Arcb., 1902, vol. xxv. p. 398. 



506 THE URINE. 

pointed out that the substance which is precipitated in this manner 
is largely a mixture of fibrinogen (fibrinoglobulin) and euglobulin. 
Nucleo-albumin may be present at the same time, but it is rare, and 
its quantity in comparison to the two albumins mentioned insignifi- 
cant. 

To demonstrate the presence of nucleo-albumin, it is necessary to 
salt out the albumins with ammonium sulphate (half saturation is 
sufficient), and then to ascertain whether any precipitation occurs 
within the limits of precipitation of nucleo-albumins. Matsumoto 
gives these as 0.1 to 0.8 (lower limit) and 1.6 and 2.2 (upper limit). 
Its limits of precipitation are the lowest of the known albumins. 1 

Whether or not Ott's test 2 in the light of this work can still be 
relied upon as a test for the demonstration of nucleo-albumin may 
be questioned. It is conducted as follows : A few cubic centimeters 
of urine are treated with an equal volume of a saturated solution of 
common salt, when Almen's solution, which consists of 5 grammes 
of tannic acid, 10 c.c. of a 25 per cent, solution of acetic acid, and 
240 c.c. of 40 to 50 per cent, alcohol, is slowly added. The 
development of a precipitate was regarded as evidence of the 
presence of nucleo-albumin. 

In order to remove nucleo-albumin from the urine, this is treated 
with neutral lead acetate, an excess of the reagent being carefully 
avoided. If it is desired to test for peptones, the filtrate is then 
treated with hydrochloric acid and the process continued as described 
above. 

Test for Haemoglobin. — The diagnosis of hemoglobinuria is based 
upon the demonstration of haemoglobin, viz., methemoglobin, in the 
urine in solution, in the absence of red corpuscles, or at least in the 
presence of only a very small number, so that an examination in the 
latter direction is also an important factor. 

Bloody urine is generally turbid, and may vary in color from 
bright red to almost black. 

Oxyhemoglobin, as such, can only be recognized by the spectro- 
scope ; it gives rise to the appearance of two bands of absorption, 
situated between D and E, as described in the chapter on the Blood. 

The urine to be examined spectroscopically should be rendered 
feebly acid by means of acetic acid, and placed before the open slit 
of the spectroscope in a test-tube, beaker, or similar vessel, when the 
two bands of oxyhemoglobin will be seen, either at once or upon 
carefully diluting with distilled water. If ammonium sulphide is 
now added, the spectrum of reduced hemoglobin will be obtained. 
It must be remembered, however, that more commonly the spectrum 
of methemoglobin is seen in cases of hemoglobinuria. 

1 Limit of precipitation of fibrinogen, 1.5 : 1.7-2.5 : 2.7 ; of fibrinoglobulin, 2.2 : 2.9; 
of euglobulin, 2.8 : 3.3; of pseudoglobulin, 3.4 : 4.6. 

2 A. Ott, Centralbl. f. inn. Med., 1895, vol. xvi. p. 38. 



ALBUMINS. 507 

The following tests, which will also indicate the presence of blood 
coloring-matter, cannot be employed to decide the nature of the 
pigment present, as methsemoglobin and oxyhemoglobin will both 
react in the same manner. 

Heller's Test. 1 — A small amount of the urine, or still better a 
portion of the sediment, is made strongly alkaline with sodium hy- 
drate and boiled. On standing, a deposit of basic phosphates forms, 
which in the presence of blood coloring-matter presents a bright-red 
color. This is referable to the formation of haBinochroinogen, as may 
be shown by spectroscopic examination. Thus controlled, the test is 
extremely sensitive, and still yields a positive result when the chem- 
ical test alone leaves one in doubt. 2 The deciding band is the first 
between D and E. Care should be had, however, that the solution 
is cold, as otherwise the hsemochromogen is transformed into haematin 
in alkaline solution. At times, when the urine contains a large 
amount of coloring-matter (bile-pigment, etc.), it may be difficult to 
determine the exact color of the sediment. In such cases the sub- 
sequent examination with the spectroscope, — the lensless instrument 
of Hering or that of Browning suffices, — is invaluable. In the 
absence of such apparatus the procedure of v. Jaksch may be em- 
ployed. To this end, the phosphatic deposit is filtered off and dis- 
solved in acetic acid, when if blood-pigment is present the solution 
becomes red, the color gradually vanishing upon exposure to the air. 
The delicacy of the test is such that oxyhemoglobin can still be 
demonstrated in a dilution of 1 : 4000. 

The Guaiacum Test. 3 — A mixture of equal parts of tincture of 
guaiacum and oil of turpentine (which has been ozonized by expos- 
ure to the air) is allowed to flow slowly along the side of a test- 
tube upon the urine to be examined, in such a manner as to form a 
distinct layer above the urine. In the presence of blood-pigment a 
white ring, which gradually turns blue, will be seen to form at the 
zone of contact. 

Doxogaxy's Test. 4 — About 10 c.c. of urine are treated with 1 
c.c. of a solution of ammonium sulphide and the same amount of 
pyridin, when in the presence of blood a more or less intense orange 
color develops, especially if looked at from above, against a white 
background. In doubtful cases the examination is to be controlled 
by a spectroscopic examination of the resulting mixture. If blood- 
pigment is present, the spectrum of hsemochromogen is obtained. 
Should the ammonium sulphide and pyridin be old, a green or brown 
color is imparted to the urine, Avhich changes to yellow upon the 
addition of ammonium hydrate. 

1 J. F. Heller, Zeit. d. K. K. Gesellsch. d. Aerzte zu Wien, 1858, No. 48. 

2 V. Arnold, Berlin. Mm. Woch., 1898, p. 283. 

3 Alnien, see Hammarsten, Lehrbuch der physiol. Chern., 3d ed. p. 488. 

4 Z. Donogany, " Darstellung d. Htemochromogen als Eeaction emf Blut," etc., Vir- 
chow's Archiv, vol. cxlviii. p. 234. 



508 THE URINE. 

Test for Fibrin. — Fibrin usually occurs in the urine in the form 
of distinct clots, the nature of which may be determined by thor- 
oughly washing with water, when they are dissolved by boiling in a 
1 per cent, solution of soda or a 5 per cent, solution of hydrochloric 
acid. On cooling, this solution is tested as for serum-albumin. 

Test for Histon. — The urine of twenty-four hours is first examined 
for albumin, and this removed if present. It is then precipitated 
with 94 per cent, alcohol, the precipitate washed with hot alcohol 
and dissolved in boiling water. Upon cooling, the solution thus 
obtained is acidified with hydrochloric acid and allowed to stand for 
several hours. During this time a cloudiness, referable to a large 
extent to uric acid, develops, which is filtered off, and the filtrate is 
precipitated with ammonia. The precipitate is collected on a small 
filter and washed with ammoniacal water until the washings no 
longer give the biuret reaction. It is then dissolved in dilute acetic 
acid and the solution tested Avith the biuret test ; if this yields a 
positive result, and if coagulation occurs upon the application of 
heat, the coagulum being soluble in mineral acids, the presence of 
histon may be inferred. 

CARBOHYDRATES. 

The carbohydrates which may occur in the urine are glucose, lac- 
tose, maltose, dextrin, levulose, certain pentoses, and animal gum. 

Glucose. — Through the researches of Wedenski, v. Udranszky, 
and others, 1 we know that traces of glucose may be encountered in 
the urine under strictly normal conditions. The amount, however, 
is extremely small, and special methods are necessary in order to 
demonstrate its presence. With the usual clinical tests normal urine 
is apparently free from sugar unless unduly large amounts have 
recently been ingested. In that event a certain amount of glucose 
is eliminated in the urine, constituting the so-called digestive gluco- 
suria of Claude Bernard. 2 

The normal limit to the assimilation of glucose on the part of the 
body economy is subject to considerable variation. Some observers 
thus report that the ingestion of such large amounts as 200 and 250 
grammes does not lead to glucosuria, while others have found sugar 
in the urine after the administration of 100 grammes. In view of 
the possible relation existing between diabetes and a lowered limit 
to the assimilation of glucose in apparently normal individuals, or at 
least in persons in whose urine glucose cannot be constantly demon- 
strated, this question has created much interest within the last 
few years and has called forth a large amount of work. The major- 

1 A. Baurnann, Ber. d. Deutsch. chem. Ges., 1886, vol. xix. p. 3218. N. Wedenski, 
Zeit. f. physiol. Chem., 1889, vol. xiii. p. 122. K. Baisch, Ibid., 1894, vol. xviii. p. 193, 
and 1895, vol. xix. p. 348. 

2 Claude Bernard, Compt. rend, de l'Aead. des Sci.. 1859, vol. xlviii. p. 673. 



CARBOHYDRATES. 509 

ity of investigators are now in accord in regarding as abnormal a 
glucosuria that follows the ingestion of 100 grammes of chemically 
pure glucose. 

The method usually employed in order to ascertain the power of 
assimilation for glucose on the part of an individual is the following : 

The patient receives 100 grammes of glucose, in substance, dis- 
solved in 500 c.c. of water, on ari empty stomach, and is instructed 
to pass his w T ater hourly during the following four to five hours. 
During this time, moreover, no food is to be taken. The individual 
specimens, as well as the urine which has been passed during the 
night, are then tested with Trommer's and Ny lander's tests, with the 
fermentation test, and with phenyl-hydrazin. A positive result, how- 
ever, is recorded only when sugar can be demonstrated with the fer- 
mentation test. 

Cane-sugar and larger amounts of glucose have also been used ; 
but it is better, on the whole, as Strauss has pointed out, to give 
glucose, and not to exceed the dose of 100 grammes. 

Especially interesting are the results which have been obtained in 
various diseases of the liver, to which organ the important function 
of preventing an undue accumulation of sugar in the blood has been 
repeatedly ascribed. Bierens de Haen l thus reports that of twenty- 
nine cases of various hepatic diseases he found sugar in eighteen 
after the administration of 150 grammes of cane-sugar; and v. 
Jaksch 2 claims to have obtained positive results in fifteen cases of 
phosphorus poisoning out of forty-three. Strauss, 3 on the other 
hand, states that he found sugar in only two of his thirty-eight 
cases, and has collected one hundred and seven additional cases from 
the literature, in only fourteen of which could sugar be demon- 
strated. If we add these together, we have one hundred and forty- 
five cases of various hepatic diseases, with negative results in 88.9 
per cent. Referring to the contradictory results obtained, Strauss 
points out that these may have been accidental in part, but that 
the interpretation which has been offered by v. Jaksch and de 
Haen may not have been correct. It is thus possible that in his 
cases of phosphorus poisoning other factors besides the changes in 
the liver, such as the action of the poison upon the nervous system, 
etc., played a role, as a digestive glucosuria may also occur in con- 
nection with other forms of intoxication, as in fevers, following the 
administration of large doses of diuretin, in acute alcoholism, etc., 
in which the liver is not the only organ that is involved. Strauss 
further shows that great care must be exercised in the selection of 
the material for such investigations, and believes that errors referable 
to this source may have been incurred by Bierens de Haen. He thus 

1 J. C, Bierens de Haen, " Ueber alimentare Glycosurie bei Leberkranken," Arch, 
f. Verdauungskrank., vol. iv. p. 4. 

2 v. Jaksch, "Alimentare Glycosurie," Prag. med. Woch., 1895, Nos. 27, 31, and 32. 

3 H. Strauss, " Leber und Glycosurie," Berlin, klin. Woch., 1898, p. 1122. 



510 THE URINE. 

cites two cases of hypertrophic cirrhosis, associated with delirium 
tremens, in which small amounts of sugar could be demonstrated in 
the urine a few days after recovery from the delirium, while shortly 
after negative results only could be obtained. The lowering effect 
of alcoholism upon the limit to the assimilation of glucose is a well- 
known phenomenon, and it would be erroneous to conclude that 
because alcoholism may call forth organic changes in the liver the 
digestive glucosuria in such cases is referable to such alterations. 
Without entering further into the question at this place, it appears 
that diseases of the liver per se do not materially lessen the as- 
similation of glucose, and that other forces are at the disposal of 
the body to supply the glycogen-forming or retaining power of the 
liver when this becomes insufficient, and that these also must be at 
fault when a digestive glucosuria is observed in association with 
hepatic disorders. 

The association of digestive glucosuria with various diseases of 
the nervous system has been carefully studied by v. Jaksch, 1 
Strumpell, H. Strauss, 2 von Oordt, Geelvink, and Arndt. 3 From 
the work of these investigators it appears that digestive glucosuria 
is rarely seen in spinal diseases, and is decidedly more common in 
functional diseases of the central nervous system than in organic 
affections. Of thirty cases of tabes examined by Strauss, digestive 
glucosuria resulted in only one after the administration of 100 
grammes of glucose, and in that one case a family history of diabetes 
existed. In sixteen further cases examined by J. Strauss negative 
results were obtained. In the neuroses a positive result was noted 
in forty-two out of two hundred and ten cases which I have been 
able to collect from the literature. Most frequently it is met with in 
the traumatic neuroses, in which Strauss observed the phenomenon 
in 37.5 per cent, of his forty cases ; while in the non-traumatic 
forms only 14.4 per cent, were insufficient in this respect. Of the 
organic diseases of the central nervous system, it appears that diffuse 
cerebral lesions referable to alcohol and syphilis are more likely to 
give rise to this form of glucosuria than the more localized lesions. 
In general paresis digestive glucosuria is thus not uncommon (H. 
Strauss, Arndt), but it is only possible to draw definite deductions 
from the study of a large amount of clinical material. Small series 
like that of J. Strauss do not give a proper idea of actual conditions, 
as he, for example, obtained negative results in all of 10 cases. 

In his examination of 5 cases of idiocy and 23 cases of imbecility, 
J. Strauss obtained positive results in only 2 of the imbeciles after 
the administration of 100 grammes of glucose; in both of the posi- 

1 v. Jaksch, loc. cit. 

2 H. Strauss, " Zur Lehre v. d. neurogenen u. d. thyreogenen Glycosurie," Deutsch. 
med. Woch., 1897, pp. 275 and 309. 

3 M. Arndt, " Ueber alimentare Glycosurie bei Neuropsychosen," Berlin, klin. 
Wocb., 1898, p. 1085. 



CARBOHYDRATES. 511 

tive cases the glucosuria was transitory and associated with the 
existence of nervous excitability. Bergenthal observed alimentary 
glucosuria in 6 cases out of 20. 

In Basedow's disease digestive glucosuria has also been noted in 
a large number of cases by Chvostek, Kraus and Ludwig, Strauss, 
Goldschmidt and Stern. Especially interesting in this connection is 
the fact that digestive glucosuria may be induced by the administra- 
tion of thyroid extract, viz., thyroidin or iodothyrin in apparently 
normal persons. Bettmann 1 thus noted glucosuria after the inges- 
tion of 100 grammes of glucose in 12 of 25 healthy individuals who 
had been treated for a week with the products in question. 

A digestive glucosuria is further observed in numerous febrile dis- 
eases, such as pneumonia, typhoid fever, acute articular rheumatism, 
scarlatina, tonsillitis, etc. The amount of sugar usually found varies 
from 0.5 to 3 per cent. ; larger amounts may, however, also be 
encountered, and one case is on record in which 8 per cent, was 
present. 2 

Very common also, as I have indicated, is the digestive glucosuria 
of drinkers, and there can be little doubt that the habitual ingestion 
of large quantities of beer and spirits will in the course of time 
lead to a more than temporary enfeeblement of the carbohydrate 
metabolism. In the course of his investigations in this direction, 
Krehl 3 found that among the Jena students the proportion of those 
in whose urine sugar appeared apparently varied with different kinds 
of beer, but was much greater after morning drinking. Of fourteen 
who drank bock or export beer in the morning, five had glucosuria. 
After the evening drinking, amounting in one case to 7 liters, of 
nineteen only one had sugar in the urine, and with Bavarian beer 
one of eleven. 

Of diseases of the skin, digestive glucosuria is notably associated 
with psoriasis ; and it is interesting to note that the same disease is 
not infrequently seen in diabetic patients. Gross thus records five 
cases, in four of which the psoriasis had existed for many years 
before the appearance of diabetic symptoms. Similar instances are 
recorded by Strauss, Grube, Polotebuoff, Nielssen, Schiitz, and others. 
Nagelschmidt 4 was able to produce glucosuria by the ingestion of 
100 grammes of glucose in eight cases out of twenty -five. 

During pregnancy digestive glucosuria is also frequently observed, 
and is by some regarded as a fairly constant symptom and of diag- 
nostic importance. The amount is variable, and while Lanz 5 records 

1 Bettmann, " Ueber d. Einfluss d. Schilddiisenbehandl. auf d. Koblendydratstoff- 
wechsel," Berlin, klin. Woch., 1897, p. 518. 

2 E. v. Bleiweis, " Ueber alimentare Glycosurie e saccharo bei acuten, fieberbaften 
Infektionskrankbeiten," Centralbl. f. inn. Med., 1900, No. 2. 

3 Krebl, "Alimentare Glycosurie nacb Biergenuss," Centralbl. f. inn. Med., 1897, 
No. 40. 

* Nagelschmidt, "Psoriasis und Glycosurie," Berlin, klin. Wocb., 1900, No. 2. 
5 Lanz, Wien. med. Presse, 1895, vol. xxxvi. 



512 THE URINE. 

one case in which 29.6 grammes of glucose were found after the 
ingestion of 100 grammes, such figures are certainly uncommon, 
and as a general rule less than 3 grammes are recovered from the 
urine. After delivery the power of assimilation for glucose no 
longer appears to be subnormal. 

A digestive glucosuria has further been observed in acute and 
chronic lead poisoning, poisoning with uitrobenzol, anilin dyes, 
opium, atropin, and carbon monoxide ; in the early stages (the first 
twelve days) of acute phosphorus poisoning ; in the febrile form of 
embarras gastrique, etc. In these conditions, however, the phenome- 
non has received little attention. 

In patients afflicted with disease of the heart, liver, and kidneys 
Gobbi l observed a digestive glucosuria, after the ingestion of from 
100 to 200 grammes of glucose, if diuretin was at the same time 
administered. 

Very important is the fact that in diabetes mellitus the sugar may 
at times disappear from the urine, while its elimination is replaced 
by an excessive excretion of uric acid or phosphates. In such cases 
glucosuria may be produced with ease by the ingestion of 100 
grammes of glucose, a point which may be of value in diagnosis. 
The exhibition of such amounts of sugar in true diabetes while 
glucosuria already exists will cause an increased elimination, while 
this apparently does not occur in other forms of glucosuria. Inter- 
esting further is the fact that in diabetic patients an increased elim- 
ination of sugar can be produced by the administration of full doses 
of copaiba. That this drug is in itself capable of lowering the limit 
to the assimilation of glucose has recently been shown by Bettmann. 
A digestive glucosuria was thus produced in four patients out of 
twelve to whom copaiba had been given for one week in amounts 
varying from 1 to 2 grammes. 

The digestive glucosuria to which reference has been made in the 
preceding pages is generally spoken of as the digestive glucosuria e 
saccharo. Similar results have been obtained after the administra- 
tion of starches in excess, viz., 150—200 grammes. But while a 
digestive glucosuria e saccharo is regarded only as a possible indica- 
tion of a pathological alteration of the carbohydrate metabolism, 
it is generally thought that every glucosuria ex amylo 1 is indicative 
of a definite disturbance in the sense of diabetes, unless special 
factors, such as an increase of the surrounding temperature, dimin- 
ished radiation of heat, or complete lack of muscular activity, are 
active. Strauss, however, has shown that in cases in which a some- 
what more than temporary predisposition toward glucosuria e sac- 
charo exists, as in alcoholics, for example, a coincident tendency 
toward glucosuria ex amylo may likewise be demonstrated. As a 

1 G. Gobbi, " La glucosuria da diuretina," II Policlinico, 1900, No. 5. 

2 E. Kiilz, Beitrage zur Pathol, u. Therap. d. Diabetes, Marburg, 1874, vol. i. p. 110. 



CARBOHYDRATES. 513 

result of his experiments he concludes that the difference between 
the digestive glucosuria e saccharo and glucosuria ex amylo is essen- 
tially a question of degree. Cceteris paribus, it appears that harm- 
ful influences of a slight character lead to glucosuria e saccharo, 
while grave insults call forth glucosuria ex amylo. It results prac- 
tically that the prognosis in those cases in which digestive glucosuria 
follows a temporary insult is far better than when the carbohydrate 
metabolism is permanently damaged, and especially when a gluco- 
suria ex amylo accompanies a glucosuria e saccharo. In the first 
instance it is scarcely likely that true diabetes will develop in the 
course of time, while in the latter this is at least possible. 

Aside from the digestive form of glucosuria which has just been 
considered, and which is produced artificially, an idiopathic transi- 
tory form is also known to occur. A transitory glucosuria, appar- 
ently of central origin, is thus noted in connection with lesions 
affecting the central as well as the peripheral nervous system, such 
as tumors and hemorrhages at the base of the brain, lesions of the 
floor of the fourth ventricle, cerebral and spinal meningitis, concus- 
sion of the brain, fracture of the cervical vertebrae, tetanus, sciatica ; 
following epileptic, hystero-epileptic, and apoplectic seizures, mental 
shock produced by railroad accidents (traumatic neuroses), etc. ; 
mental strain and worry, fatigue, and anxiety. Glucosuria follow- 
ing epileptic and apoplectic attacks, however, does not appear to be 
so common as is generally believed, v. Jaksch was unable to de- 
monstrate the presence of sugar in fifty recent cases of hemiplegia, 
and in a large number of cases of epilepsy, with urines voided 
within the first few hours following the seizure I have reached only 
negative results. 

In Basedow's disease transitory glucosuria may also occur, and 
it is well established that a relation may exist between the disease 
in question and the complex of symptoms is designated as diabetes 
mellitus. 1 

Siegmund noted a transitory glucosuria in 52.38 per cent, of 
general paretics, in 7.4 per cent, of epileptics, and in 3.77 per cent, 
of dementia cases, while it was not observed in other mental dis- 
eases. In reference to the post-epileptic glucosuria which has been 
noted by some of the older observers more especially, an analysis of 
their work has led me to the conclusion that their inferences were 
scarcely justifiable, as a wholly satisfactory proof of the presence of 
sugar has not been furnished. 2 

In cases of cholelithiasis, contrary to what has been maintained 
by one or two observers, glucosuria is unusual. 

1 Dumontpallier, "Goiter exophthalinique et glycosurie," Compt. rend. d. 1. soc. 
d. Biol., 1867. O'Neill, "Exophthalmic Goitre and Diabetes occurring in the same 
Person," Lancet, 1878, Pt. 1. p. 9. S. Bettmann, Munch, med. Woch., 1896, vol. xliii. 
Nos. 49 and 50. E. Grawitz, Fortsch. d. Med., 1897, vol. xv, K. Osterwald, Inaug. 
Diss., Gottingen, 1898. H. Stern, Jour. Am. Med. Assoc, 1902, vol. xxxix. p. 972. 

2 See, also, Araki, Zeit. f. phys. Chem., vol. xv. p. 363. 

33 



514 THE URINE. 

It is well known that Claude Bernard experimentally produced 
a transitory glucosuria by puncturing a certain spot in the floor 
of the fourth ventricle, the supposed origin of the hepatic vaso- 
motor nerves, and it is not improbable that this neurotic form 
of glucosuria is due to some direct or reflex influence affecting that 
portion of the medulla. 

The transitory glucosuria occasionally observed in acute febrile 
diseases, such as typhoid fever, scarlatina, measles, cholera, diph- 
theria, influenza, and especially malaria, particularly during conva- 
lescence, may possibly be referable to the action of ptomams or 
leukomams upon this centre. Seegen reports five cases of malaria 
with " diabetes " in which both conditions disappeared under the 
administration of quinin. In diphtheria glucosuria appears to be 
of common occurrence. Binet thus obtained a positive result in 
twenty -nine cases out of seventy ; twenty-seven times in severe in- 
fections out of thirty-eight, and twice in mild cases out of thirty- 
two. I have personally found a transitory glucosuria in four cases 
out of thirty-two ; the infection in these was of moderate severity. 
Hibbard and Morrissey arrived at similar results. 1 

A glucosuria of toxic origin has been noted in cases of poisoning 
with curare, chloral hydrate, sulphuric acid, arsenic, alcohol, carbon 
monoxide, morphin, etc., and even after simple transfusion of nor- 
mal salt solution into the blood. Phloridzin, a glucoside obtained 
from the bark of the root of the apple tree, will likewise cause sugar 
to appear in the urine. The glucosuria thus produced is, however, 
only temporary, and ceases upon withdrawal of the drug. 2 Of 
interest is the glucosuria which occasionally follows the administra- 
tion of thyroid extract or of iodothyrin, as there is evidence to 
show that in such cases a special predisposition to glucosuria exists. 
When carried to an extreme degree true diabetes may develop, which 
subsequently cannot be arrested by withdrawal of the substance. 3 

The occurrence of a transitory glucosuria under the conditions 
above mentioned, and which may be met with in almost any disease, 
moreover, while interesting from a theoretical standpoint, must in 
the majority of instances be regarded as a medical curiosity only, 
and it is but rarely possible to draw either diagnostic, prognostic, or 
therapeutic conclusions from its existence. 

A persistent form of glucosuria is noted in connection with certain 
lesions of the brain, especially those affecting the floor of the fourth 
ventricle, and is at times of considerable value in diagnosis. This 
is also observed after removal of the thyroid gland, and in cases in 
which thyroid extract has been administered in unduly large amount. 

1 C. M. Hibbard and M. J. Morrissey, " Glycosuria in Diphtheria," Jour. Exper. 
Med., vol. iv. p. 137. 

2 Zuntz, " Zur Kenntniss d. Phloridzindiabetes," Du Bois' Archiv, 1895, p. 570. 

3 H. Strauss, " Neurogene and thyreogene Glucosurie," Deutsch. med, Woch., 1897, 
Nos. 19 and 20, 



CARBOHYDRA TES. 515 

A continuous elimination of sugar, however, is noted principally 
in the complex of symptoms to which the term diabetes mellitus has 
been applied. 

Diabetes mellitus is essentially a persistent form of glucosuria 
associated with the occurrence of a more or less intense polyuria and 
a greatly increased elimination of all the metabolic products normally 
found in the urine, with the exception of uric acid, which is usually 
present in diminished amount. In the more advanced cases aceto- 
nuria, lipuria, and lipaciduria may also exist. Diabetes, however, is 
not a persistent form of glucosuria in an absolute sense of the word, 
as periods may occur in the course of the disease when glucose is 
temporarily absent. 

The quantity of sugar excreted may be very large, and 180 to 360 
grammes pro die are amounts which may be frequently observed. 
This quantity may diminish to zero under various conditions, such 
as the occurrence of intercurrent diseases, but often also without any 
apparent cause, and not infrequently in the condition which has been 
termed diabetic coma. Cases are also observed in which from begin- 
ning to end mere traces are eliminated, the total amount of sugar 
not exceeding a few grammes, while the course of the disease rapidly 
tends toward a fatal termination, so that the severity of the pathological 
process cannot he measured by the amount of sugar eliminated. A 
few years ago I had occasion to observe a diabetic patient in whom 
for months a daily examination of the urine never revealed the 
presence of more than 5 to 10 grammes of sugar, and in whom 
death occurred after eighteen months. 

At the same time it should be remembered that diabetes cannot 
be excluded by one or even more negative urinary examinations, and 
the value of repeating such examinations three or four hours after 
the exhibition of 100 grammes of glucose, as indicated, cannot be 
too strongly urged. 

Clinicians are in the habit of determining the severity of a case, 
to a certain extent at least, from the condition of the urine under a diet 
free from starches and sugars, and generally regard those cases as the 
more serious in which the glucosuria does not disappear under a diet 
of this character, while a more favorable prognosis is given if the 
sugar disappears. It should be remembered, however, that there 
are numerous exceptions to this rule, and that a light case, — i. e., 
one in which the sugar disappears under appropriate dietetic treat- 
ment, — may suddenly exhibit symptoms seen only in the most 
severe forms, or succumb to one of the numerous intercurrent 
maladies, while apparently severe cases may assume the more benign 
type. 

It may not be out of place in this connection to say a few words 
regarding the specific gravity of the urine. While usually very high, 
varying between 1,030 and 1.060, as pointed out in the chapter 



516 THE URINE. 

on Specific Gravity, comparatively low figures are noted at times, 
such as 1.012, corresponding to a quantity of urine not exceed- 
ing 1000 c.c, and implying, of course, a diminished elimination 
of solids. This is especially marked in those cases described by 
Hirschfeld, 1 in which, as pointed out in the chapter on Urea, the 
resorption of nitrogenous material from the digestive tract is below 
the normal. Polyuria, a fairly constant symptom of the more com- 
mon types of diabetes mellitus, is much less pronounced in Hirsch- 
feld' s form, and may be altogether absent, although it is true that 
this may occur in ordinary diabetes also. 

The simultaneous occurrence of glucosuria, acetonuria, lipuria, 
and lipaciduria. (which see) is probably always indicative of true 
diabetes. 

It is, of course, impossible to enter here into a detailed considera- 
tion of the origin of diabetes. Suffice it to say that a persistent glu- 
cosuria, aside from nervous influences, may be referable, on the one 
hand, to an inability on the part of the liver to transform into gly- 
cogen all of the sugar which is carried to this organ ; or, on the other 
hand, to an inability on the part of the muscular system of the body 
to utilize all the sugar sent to it. Accordingly, we may distinguish 
between a hepatogenic and a myogenic diabetes. As a matter of fact, 
cases are seen, usually belonging to the milder form of the disease, 
in which the sugar may be temporarily caused to disappear from the 
urine by muscular exercise. On the other hand, again, cases are 
seen, and unfortunately only too frequently, in which, notwithstand- 
ing a total abstinence from carbohydrates and a free indulgence in 
muscular exercise, the sugar does not disappear from the urine. In 
such cases it is permissible to speak of a hepatogenic combined with 
a myogenic diabetes. 

Within recent years it has been shown that pancreatic disease is 
frequently associated with diabetes, and while the number of cases 
in which no pancreatic lesions are discovered is still too large to war- 
rant the conclusion that disease of this organ is invariably associated 
with glucosuria, it still must be admitted that lesions of the pancreas 
are the more frequently met with in diabetes the more carefully the 
organ is examined. So much appears to be certain, that diabetes 
may be produced by pancreatic disease. As to the manner, how- 
ever, in which such a result can occur we are in ignorance. In 
this connection it is interesting to note that, according to Opie, dis- 
ease of the areas of Langerhans more especially is associated with 
the clinical picture of diabetes, while lesions affecting the secreting 
portion of the gland only do not influence the carbohydrate metab- 
olism. 2 These observations of Opie have been largely confirmed by 
other observers. 

1 F. Hirschfeld, " Ueber eine neue klinische Form d. Diabetes," Zeit. f. klin. Med., 
vol. xix. pp. 294 and 325. 

2 Opie, Jour. Exper. Med., 1901, vol. v. p. 527. 



CARBOHYDRATES. 517 

Hirschfeld pointed out the fact that while in the majority of 
diabetic patients the proteid food ingested is quite satisfactorily 
utilized, the assimilation of fats and albumins is much below nor- 
mal in others, and particularly so in cases of diabetes associated with 
pancreatic disease (see also Urea). Observations in this direction 
are as yet very scanty, so that a definite opinion cannot be expressed 
regarding the utility in diagnosis of investigations similar to those of 
Hirschfeld. I have had occasion to observe a diabetic patient for 
some time in whom, notwithstanding that conclusions were reached 
similar to those of Hirschfeld, the existence of pancreatic disease 
could not be determined post mortem. 

Whether or not a renal and a thyroigenic diabetes also exists, as 
has recently been suggested, remains an open question. 1 That Base- 
dow's disease may be associated with diabetes mellitus I have already 
pointed out. 

Tests for Sugar. — The tests for sugar usually employed in the 
clinical laboratory depend upon the following properties of sugar : 

1. In the presence of alkalies it acts as a reducing agent upon 
certain metallic oxides, such as those of copper and bismuth (Feh- 
ling's, Trommer's, Bottger's, and Xylander's tests). 

2. In the presence of yeast (Saccharomyces cerevisia?) it under- 
goes fermentation, with the formation of alcohol, carbonic acid, 
succinic acid, glycerin, and a number of other bodies, such as amyl 
alcohol, etc. (fermentation test). 

3. With phenylhydrazin sugar forms an insoluble crystalline 
compound — phenylglucosazon. 

4. Solutions of glucose turn the plane of polarized light to the 
right, from which property glucose has also received the name 
dextrose. 

In every case the urine should first be tested for the presence of 
albumin, which should be removed by boiling. 

Trommer's Test. 2 — A few cubic centimeters of urine are strongly 
alkalinized with sodium hydrate solution, and treated with a 5 per 
cent, solution of cupric sulphate, added drop by drop, until the 
cupric oxide formed is no longer dissolved. The mixture is care- 
fully heated, when in the presence of sugar a yellow precipitate of 
cuprous hydroxide is formed, which gradually settles to the bottom 
as a sediment of red cuprous oxide. 

It is important to note that while sugar, unless present in mere 
traces, can readily be detected in this manner, other substances are 

1 Diabetes : J. Seegen, Die Zuckerbildung im Thierkorper, Berlin, 1890, p. 260. 
v. Xoorden, Pathol, d. Stoffwechsels, Berlin, 1893. Seegen, " Ueber d. Zuckergehalt d. 
Blutes von Diabetikern." Wien. med. Woch., 1886, Nos. 47 and 48. F. W. Pavy, 
" Ueber die Bebandlung von Diabetes meilitus," Verhandl. d. X. Interuat. Med. 
Congr., 1891, II., Abt. 5, p. 80. P. F. Eichter, '" Xierendiabetes," Deutsch. med. 
Woch., 1899, p. 840. 

2 C. Trommer, Annal. d. Ckem. u. Pharm., 1841, vol. xxxix. p. 361. 



518 THE URINE. 

or may be present in the urine, such as uric acid, kreatin and krea- 
tinin, allantoin, nucleo-albuniin, milk-sugar, pyrocatechin, hydro- 
chinon, and bile-pigment, which likewise reduce cupric oxide. 
Following the ingestion of benzoic acid, salicylic acid, glycerin, 
chloral, sulphonal, etc., reducing substances also appear. These may 
generally be disregarded, it is true, if care is taken not to boil the 
urine after the addition of the cupric sulphate, as the precipitation 
of cuprous oxide in the presence of sugar takes place before this point 
is reached. Unfortunately, however, the test when thus applied 
yields negative results, or results which are doubtful, if traces only 
are present, so that it cannot be utilized, as a rule, in the study of 
transitory or digestive glucosuria. 

Feheing's Test. 1 — This is a modification of the test just described, 
and can be recommended only with the same restrictions. 

Two solutions are employed, which must be kept in separate 
bottles, the one containing 34.64 grammes of crystallized cupric sul- 
phate, dissolved in 500 c.c. of distilled water, and the other 173 
grammes of potassium and sodium tartrate and 125 grammes of 
potassium hydrate, dissolved in an equal volume of water. Equal 
parts of the two solutions, mixed in a test-tube and diluted with 
four times as much water, are boiled, when a small amount of urine 
is added. In the presence of sugar a precipitate of the yellow 
hydroxide of copper or of red cuprous oxide will be produced ; but 
care should be taken only to warm, and not to boil the solution after 
addition of the urine. 

Not infrequently it will be observed that upon standing, when no 
precipitation has occurred previously, the blue color of the mixture 
changes to an emerald green, while the solution at the same time 
becomes turbid. Such a phenomenon should not be referred to the 
presence of sugar, as it is in all probability due to the action of other 
reducing substances, such as those mentioned above. 

Bottger's Test with Nylander's Modification. 2 — A few 
cubic centimeters of urine are treated with Almen's solution in the 
proportion of 11 : 1. This is prepared by dissolving 4 grammes 
of potassium and sodium tartrate, 2 grammes of bismuth subnitrate, 
and 10 grammes of sodium hydrate in 90 c.c. of water, heating the 
solution to the boiling-point and filtering upon cooling, when it 
should be kept in a colored glass bottle. The mixture of urine 
and Almen's fluid is thoroughly boiled, when in the presence of 
sugar a grayish, dark-brown, and finally a black precipitate, con- 
sisting of bismuthous oxide or of metallic bismuth, is obtained. 
Albumin, if present, must first be removed, as, owing to the sulphur 
contained in the albuminous molecule, alkaline sulphides would be 
formed upon boiling, and, acting upon the bismuth, give rise to the 

1 H. Fehling, Annal. d. Chem. u. Pharm., 1849, vol. lxxii. p. 106. 

2 E. Nylander, Zeit. f. physiol. Chem., 1883, vol. viii. p. 175. 



CARBOHYDRATES. 



519 



formation of black bismuth sulphide, which might be mistaken for 
metallic bismuth. Rhubarb -pigment, as well as melanin and melan- 
ogen (which see), and free hydrogen sulphide must also be absent, 
as misleading results will otherwise be obtained. 

Nylander's test, as well as those of Trommer and Fehling, is, 
however, not without objections, as a partial reduction of the bis- 
muth subnitrate may be produced by other substances, such as 
kairin, tincture of eucalyptus, turpentine, and large doses of quinin. 

Fermentation Test. 1 — A small piece of ordinary compressed 
yeast is shaken with some of the suspected urine and a test-tube filled 
with the mixture, to which some mercury is added. The tube is then 
inverted into a vessel containing mercury, and allowed to stand in 
a warm place (22°— 28° C). If sugar is present, fermentation will 
occur in the course of twelve hours, and the carbon dioxide formed 
rise to the top of the tube, gradually displacing more and more of 
the urine or mercury as the amount of the gas increases. It is easy 
to demonstrate that the gas thus formed is carbon dioxide by 
introducing a small piece of caustic soda into the urine, when, 
owing to absorption of the carbon dioxide, the liquid will again rise 
in the tube. Very convenient for this purpose also are the saccha- 

Fig. 113. 




Einhorn's saceharimeter. 



rimetric tubes of Einhorn (Fig. 113) or Lohnstein 2 (Fig. 115), 
which are employed as just described, a little mercury being poured 



1 M. Einhorn, Virehow's Archiv, 1885, vol. cii. p. 263. 

2 Lohnstein, Berlin, klin. Woch., 1898, p. 866. 



520 THE URINE. 

into the bent limb to guard against escape of gas. As the yeast 
itself, however, may give rise to the formation of a little gas in the 
absence of sugar, it will always be well to make a control-test with 
normal urine — i. e., to prepare a similar tube with normal urine 
mixed with yeast, and to allow this to stand at the same temperature. 
If a positive result is thus obtained, there can be no doubt as to the pres- 
ence of a fermentable substance in the urine. This, however, is not 
necessarily glucose, as other carbohydrates, such as lactose, maltose, and 
levulose, may likewise undergo fermentation. Still, if large amounts 
of gas are obtained, and if Trommer's test also yields a positive result, 
it will be fairly safe to regard the substance present as glucose. 

Phenylhydrazin Test. 1 — As originally proposed by v. Jaksch, 
the test is conducted as follows : 6 to 8 c.c. of urine are treated 
with 0.4 to 0.5 gramme of phenylhydrazin hydrochlorate and 1 
gramme of sodium acetate, and warmed until the salts have been 
dissolved, a little water being added if necessary. The tube is 
placed in boiling water for twenty to thirty minutes, and then 
transferred to a beaker filled with cold water. If sugar is pres- 
ent in moderate amounts, a bright-yellow crystalline deposit will 
at once be thrown down and partly adhere to the sides of the tube. 
But even in the presence of mere traces a careful microscopical ex- 
amination will reveal the presence of crystals of phenylglucosazon 
(Plate XIX.). These are seen singly or arranged in bundles and 
sheaves composed of delicate bright-yellow needles which are insol- 
uble in water. 

Still more convenient is the following modification of the test, as 
suggested by Cipollina : 2 5 drops of pure phenylhydrazin, 0.5 c.c. 
of glacial acetic acid or 1 c.c. of 50 per cent, acetic acid are placed 
in a test-tube together with 4 c.c. of urine. The mixture is boiled 
for about one minute over a small flame, while shaking so as to avoid 
bumping as much as possible; 4 or 5 drops of sodium hydrate solution 
(specific gravity 1.16) are added, but the solution must remain acid; 
the boiling is continued for a few seconds and the mixture then 
allowed to cool. The rapidity with which the glucosazou crystals 
separate out depends somewhat upon the specific gravity of the urine. 
If this is low, they form in a few minutes, even though the amount 
of sugar does not exceed 0.05 per cent. If, on the other hand, the 
specific gravity is high, yellow balls and Stechapfelformen result, 
while typical rosettes develop only after twenty to thirty minutes, 
and at times one is even then left in doubt as to the result. If the 
urine contains more than 0.2 per cent, of sugar, even though the 
specific gravity be high, the formation of typical crystals occurs 
within a few minutes. If with this modification no crystals are 

1 v. Jaksch, Zeit, f. klin. Med,, 1886, vol. xi. p. 20. 

2 A. Cipollina, Deutsch. med. Woch., 1901, vol. xxvii. p. 334. 



PLATE XIX. 




Phenyl-Glueosazon Crystals obtained from a Diabetic Urine. 



CARBOHYDRATES. 521 

obtained at the expiration of an hour, we may infer that no sugar is 
present. 

This test, properly applied, is undoubtedly not only the most deli- 
cate, but at the same time the most reliable, as no other substances 
which may be present in the urine, excepting maltose and certain 
pentoses, will give rise to the formation of an osazon. Hence, when- 
ever doubt is felt as to the nature of a substance reacting in a posi- 
tive manner with the reagents described above, recourse should be 
had to this test. It has been stated that maltose forms an exception ; 
this, however, will never become embarrassing, as the microscopical 
appearance of the maltosazon crystals differs from that of the phenyl- 
glucosazon. The melting-point of phenylglucosazon (205° C), 
moreover, is about 15 degrees higher than that of the maltosazon — 
190°-191° C. To determine this point, it is necessary to filter 
off the osazon, and, after washing with water, to dissolve it upon a 
filter by means of a little hot alcohol. From this alcoholic solution 
it is reprecipitated by water, when it may be collected and dried over 
sulphuric acid. The melting-point is then determined according to 
the usual methods. 

The pentosazons also can be readily distinguished from glucosazon 
by their melting-points (which see). 

The amount of lactose which may be found in the urine is far too 
small to give rise to the formation of an osazon when the test is 
directly applied to the urine. 

With the conjugate glucuronates phenylhydrazin also combines to 
form crystalline compounds, but these may likewise be distinguished 
by their melting-points and the form of the crystals. Such com- 
pounds, moreover, are usually not present in amounts sufficient to 
give rise to confusion (see Glucuronic Acid). 

Polarimetric Test. — Glucose turns the plane of polarized light 
to the right, but the same may be said of maltose, the degree of 
polarization of which is even more marked, so that it may be impos- 
sible to state in a given case whether such rotation is referable to a 
large quantity of glucose or to a smaller quantity of maltose. The 
latter substance, however, occurs in the urine but rarely, and may be 
recognized not only by the microscopical appearance of its osazon, 
but also by the fact that its power of reduction is increased in the 
presence of sulphuric acid and by the application of heat. 

An error which may further arise with the employment of the 
polarimetric method is referable to the fact that if glucose is pres- 
ent in only small amounts, while the urine contains large quantities 
of ^-oxybutyric acid, the latter turning the plane of polarized light 
to the left, it may happen that the rotation in this direction will neu- 
tralize or even counterbalance any rotation to the right which may 
be due to glucose. In such cases, however, the urine will react in a 



522 



THE URINE. 



positive manner with the other reagents described, and the fermented 
urine will, moreover, turn the plane of polarization still more strongly 
to the left, indicating the presence of a dextrorotatory substance, and 
in all probability of glucose. 

The delicacy of this method varies with the instrument employed ; 
the figures given below were obtained with the apparatus of Lippich, 
which yields the best results. 

(For a description of this method see the Quantitative Estimation 
of Sugar by Means of the Polarimeter.) 



Table showing the Delicacy of the Tests described. 

Trommer's test 0.0025 per cent. 

Fehling's test 0.0008 " 

Nylander's test 0.025 " 

Fermentation test 0.1-0.05 " 

Phenylhydrazin test 0.025-0.05 " 

Polarimetric test 0.025-0.05 " 



Table showing the Behavior of the Various Forms of Sugar which 
may occur in the urine toward the tests described. 



Trommer's, viz., 
Fehling's test. 



Nylander'i 
test. 



Fermenta- 
tion test. 



Phenylhydrazin 

test. 



Polarimetric 

test. 



Glucose. 



Levulose. 



Maltose. 



Lactose. 



Laiose. 



Positive reaction. 



Positive reaction. 



Positive reaction. 



Positive reaction. 



Positive reaction 
on boiling only ; 
1.2-1.8 per cent, 
more is obtain- 
ed than by the 
polarimeter. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



Positive 
reaction. 



No re- 
action or 
only a 
very faint 
one. 



No reac- 
tion. 



Positive reaction 
melting-point 
205° C 

Same osazon ob- 
tained as with 
glucose, only 
more rapidly. 

A maltosazon is 
formed; melting- 
point 190°-191° C 

No reaction in the 
concentration in 
which it may oc- 
cur in the urine ; 
melting-point 
200° C. 

With phenylhy- 
drazin a yellow- 
ish-brown, non- 
crystallizable oil 
is obtained. 



Rotation toward 
the right. 



Rotation 
the left. 



toward 



Rotation toward 
the right. 



Rotation toward 
the right; in- 
creased by boil- 
ing with a 2.5 per 
cent, solution of 
sulphuric acid. 

No reaction, or ro- 
t a t i o n toward 
the left. 



Clinically, it is unimportant to search for minute traces of sugar, 
such as may be found in every normal urine, and the reader is 
referred to special works on physiological chemistry for a considera- 
tion of the methods generally employed (see method of Baumann 
and v. Udranszky. 

Quantitative Estimation of Sugar. — The methods used in the 
quantitative estimation of sugar are essentially based upon the quali- 
tative tests described. 



CARBOHYDRATES. 523 

Fehling's Method. 1 — Fehling's solution prepared as described 
above is of such strength that the copper contained in 10 c.c. is 
completely reduced by 0.05 gramme of glucose. If then urine is 
carefully added to this quantity until complete reduction takes place, 
the amount of sugar contained in a given specimen of urine can be 
readily calculated according to the following equation : 

y : 0.05 : : 100 : x ; and x = — , 

y 

in which y indicates the number of cubic centimeters of urine 
required to reduce the 10 c.c. of Fehling's solution, and x the amount 
of sugar contained in 100 c.c. of urine. 

As the best results are obtained only if from 5 to 10 c.c. of urine 
are used in one titration, it is usually necessary to dilute the urine 
to the required degree ; in the determination of this point the specific 
gravity may serve as a guide. As a general rule, urines of a specific 
gravity of 1.030 should be diluted five times, and if the density is 
still higher ten times. To be certain that the proper degree of 
dilution has been reached, 5 c.c. of Fehling's solution are treated 
with 1 c.c. of the diluted urine, a little caustic soda solution and 
distilled water being added to make in all about 25 c.c. This mixt- 
ure is thoroughly boiled ; if the fluid still remains blue, another 
1 c.c. of diluted urine is added, and so on, until the last two tests 
differ by 1 c.c. of urine, the last cubic centimeter added causing a 
separation of cuprous oxide. In this manner the percentage of 
sugar may be approximately determined. Albumin, if present, 
must first be removed by boiling. 

Ten c.c. of Fehling's solution diluted with 40 c.c. of water are 
placed in a porcelain dish and boiled. While boiling, the diluted 
urine is added from a burette, 0.5 c.c. at a time, when, as a rule, the 
precipitated cuprous oxide will rapidly settle, so that gradually a 
white bottom may be seen through the blue field, the color of which 
becomes less and less intense upon the further addition of urine 
until finally the solution is almost colorless. When this point is 
reached the urine is added drop by drop until the decolonization is 
complete. The degree of dilution multiplied by 5 and the result 
divided by the number of cubic centimeters of diluted urine em- 
ployed will then indicate the percentage-amount of sugar. In the 
table on page 524 the percentage results corresponding to the 
number of cubic centimeters of undiluted urine employed will be 
found. 

Unfortunately, it is difficult, as a general rule, to determine ex- 
actly the point when all the copper has been reduced — i. e., the point 
at which the blue color has entirely disappeared. When it is thought 
that this has been reached, about 1 c.c. should be filtered through 

1 Loc. cit. 



524 



THE URINE. 



thick Swedish filter-paper, and the filtrate (which must be absolutely 
clear) acidified with acetic acid and treated with a drop or two of a 
solution of potassium ferrocyanide. If unreduced copper is still 
present in the solution, a brown color will result, indicating that 
sufficient urine has not been added. But if, on the other hand, no 
brown discoloration is noted, it is possible that the desired point has 
been passed, when the titration should be repeated. At times the 
precipitate will not settle at all, and even pass through the filter, so 
that it is practically impossible to determine the end of the reaction. 
In such cases the following procedure, suggested by Cause, will be 
found of value : 

Ten c.c. of Fehling's solution are diluted with 20 c.c. of distilled 
water and treated with 4 c.c. of a 0.05 per cent, solution of potas- 
sium ferrocyanide. While boiling, the diluted urine is added drop 
by drop until the blue color entirely disappears. A precipitate does 
not form with this method. 

Sugar. — Quantity of Glucose pro liter, corresponding to the number of cubic centimeters 
used for the complete reduction of 10 cubic centimeters of Fehling's solution. 





1 


JL 


_2_ 


3 


_4_ 


5 


6 


7 


_8_ 


_9_ 






10 


10 


10 


10 


10 


10 


10 


10 ■ 


10 


1 


50.00 


45.44 


41.68 


38.46 


35.70 


33.32 


31.24 


29.40 


27.76 


26.30 


2 


25.00 


23.80 


22.72 


21.72 


20.84 


20.00 


19.22 


18.50 


17.84 


17.24 


3 


16.66 


16.00 


15.62 


15.14 


14;15 


14.28 


13.88 


13.50 


13.14 


12.82 


4 


12.50 


12.18 


11.90 


11.62 


11.36 


11.10 


10.86 


10.62 


10.40 


10.20 


5 


10.00 


9.80 


9.60 


9.42 


9.24 


9.08 


8.92 


8.76 


8.62 


8.50 


6 


8.32 


8.18 


8.06 


7.92 


7.80 


7.68 


7.56 


7.44 


7.34 


7.24 


7 


7.14 


7.04 


6.94 


6.86 


6.78 


6.66 


6.56 


6.48 


6.40 


6.32 


8 


6.24 


6.16 


6.08 


6.02 


5.94 


5.88 


5.80 


5.74 


5.68 


5.60 


9 


5.54 


5.48 


5.42 


5.36 


5.30 


5.24 


5.20 


5.16 


5.12 


5.06 


10 


5.00 


4.94 


4.90 


4.82 


4.78 


4.76 


4.70 


4.66 


4.62 


4.58 


11 


4.54 


4.50 


4.46 


4.42 


4.38 


4.34 


4.30 


4.26 


4.22 


4.20 


12 


4.16 


4.14 


4.12 


4.08 


4.04 


4.00 


3.98 


3.96 


3.92 


3.86 


13 


3.84 


3.80 


3.78 


3.76 


3.74 


3.70 


3.68 


3.66 


3.62 


3.58 


14 


3.56 


3.54 


3.52 


3.48 


3.46 


3.44 


3.42 


3.40 


3.36 


3.34 


15 


3.32 


3.32 


3.28 


3.26 


3.24 


3.22 


3.20 


3.18 


3.16 


3.14 


16 


3.12 


3.10 


3.08 


3.04 


3.04 


3.02 


3.00 


2.98 


2.96 


2.94 


17 


2.94 


2.92 


2.90 


2.88 


2.86 


2.84 


2.82 


2.82 


2.80 


2.78 


18 


2.76 


2.76 


2.74 


2.72 


2.70 


2.70 


2.68 


2.64 


2.64 


2.64 


19 


2.62 


2.62 


2.60 


2.60 


2.58 


2.56 


2.56 


2.54 


2.52 


2.52 


20 


2.50 


2.50 


2.48 


2.48 


2.44 


2.42 


2.42 


2.40 


2.40 


2.38 


21 


2.38 


2.36 


2.34 


2.34 


2.32 


2.32 


2.30 


2.30 


2.28 


2.28 


22 


2.26 


2.26 


2.24 


• 2.24 


2.22 


2.22 


2.20 


2.20 


2.18 


2.18 


23 


2.16 


2.16 


2.14 


2.14 


2.12 


2.12 


2.12 


2.10 


2.10 


2.10 


24 


2.08 


2.08 


2.06 


2.06 


2.06 


2.04 


2.04 


2.02 


2.02 


2.02 


25 


2.00 


1.98 


1.98 


1.96 


1.96 


1.96 


1.94 


1.94 


1.92 


1.92 


26 


1.92 


1.92 


1.90 


1.90 


1.88 


1.88 


1.88 


1.86 


1.86 


1.86 


27 


1.84 


1.82 


1.82 


1.82 


1.82 


1.80 


1.80 


1.80 


1.80 


1.80 


28 


1.78 


1.76 


1.74 


1.74 


1.74 


1.74 


1.74 


1.74 


1.74 


1.72 


29 


1.72 


1.70 


1.70 


1.70 


1.70 


1.68 


1.68 


1.68 


1.68 


1.66 


30 


1.66 


1.66 


1.65 


1.63 


1.63 


1.62 


1.62 


1.62 


1.62 


1.62 



In order to obtain reliable results, however, the Fehling solution 
must be prepared with great care and its strength determined. This 
may be done in the following manner : 0.2375 gramme of pure 
crystallized cane-sugar, dried at 100° C, is dissolved in 40 c.c. of 
distilled water, to which 22 drops of a 0.1 per cent, solution of sul- 
phuric acid have been added. This solution is kept on the boiling 



CAEBOHYDRA TES. 525 

water-bath for an hour, when it is allowed to cool and diluted to 
100 c.c. with distilled water. Twenty c.c. of this solution will then 
contain exactly 0.05 gramme of glucose, corresponding to 10 c.c. of 
Fehling's solution, if this is of the required strength. If too strong, 
so that 21 c.c. of the sugar solution, for example, are required to 
obtain a complete reduction of the copper, the strength of Fehling's 
solution may be determined accordiog to the equation : 20 : 0.05 : : 
21 : x; and x = 0.0525. If too weak, on the other hand, so that 
19 c.c, for example, are required, its strength is similarly deter- 
mined : 20 : 0.05 ::19:x; and x = 0.0475. 

Kxapp's Method. 1 — This method is said to be more satisfactory 
than that of Fehling. Daylight is not necessary ; the method is 
simpler, and it is applicable even iu cases in which the amount of 
sugar is small ; and the solution keeps for a long while. 

The principle of the method depends upon the observation that 
mercuric cyanide in alkaline solution is reduced to merallic mercury 
in the presence of sugar. The solution required should contain 10 
grammes of chemically pure, dry mercuric cyanide and 100 c.c. of 
a solution of sodium hydrate (sp. gr. 1.145) to the liter. Twenty 
c.c. of this solution correspond to 0.05 gramme of glucose. 

Method. — Twenty c.c. of the solution are placed in a small 
retort and diluted with 80 c.c. of water. If we have reason to 
suppose that the urine contains less than 0.5 per cent, of sugar, 40 
to 60 c.c. are sufficient. The solution is then heated to the boiling- 
point, when the diluted urine (see below) is added, at first 2 c.c. 
at a time, then 1 c.c, 0.5 c.c, 0.2 c.c, and 0.1 c.c, as the final 
point is approached. After each addition the solution is boiled for 
one-half minute. As the end-reaction is approached the solution 
clears, and the mercury, together with the phosphates, settles to the 
bottom. The final point is determined by placing a drop of the 
supernatant fluid upon a piece of clean, white Swedish filter-paper, 
and holding this first over a bottle containing concentrated hydro- 
chloric acid and then over one containing a saturated solution of 
hydrogen sulphide. If all the mercuric cyanide has not been 
reduced, a yellow spot will result, the color of which becomes the 
more manifest if it is compared with one which has not been ex- 
posed to the action of hydrogen sulphide. As soon as the mercury 
is entirely reduced the readiug is taken. 

Example. — Supposing that 15 c.c of urine have been required, the 
corresponding amount of sugar is then found according to the fol- 
lowing equation, 20 c.c of Knapp's solution requiring 0.05 gramme 
of sugar for its reduction : 

15 : 0.05 : : 100 : x ; 15a; = 5 ; and x = 0.333 per cent. 
1 K. Knapp, Annal. d. Chem. u. Pharni., 1870, vol. cliv. p. 252. 



526 THE URINE. 

Precautions : 1. Albumin must first be removed. 

2. The urine should not contain more than 0.5 to 1 per cent, of 
sugar. The urine is hence diluted, if necessary , as with Fehling's 
method. 

Differential Density Method. 1 — This method is very useful 
in clinical work, and should be preferred to the more uncertain titra- 
tion with Fehling's solution, unless considerable experience has been 
acquired with the method. 

The specific gravity of the urine is accurately ascertained by 
means of a pyknometer, or a hydrometer graduated to the fourth 
decimal and provided with a thermometer indicating tenths of a 
degree. The temperature at which the specific gravity is taken 
should be that for which the hydrometer has been constructed, the 
urine being heated or cooled to the desired degree. One hundred 
to 200 c.c. are then set aside in a flask, after the addition of some 
yeast which has been washed free from mineral material, loosely 
stoppered or provided with an arrangement like the one shown in 
the accompanying figure (Fig. 114). After twenty-four hours if 
but little sugar is present, or forty-eight hours if there is much, the 
specific gravity is again determined under the precautions given, 
after having filtered the urine. The difference in the specific gravity 
is multiplied by 230, an empirical factor which has been found by 
dividing the amount of sugar ascertained by titration or polarization 
with the difference in the density of the urine after fermentation. 
The result indicates the percentage of sugar. 

The process may be hastened if to each 100 c.c. of urine 2 
grammes of potassium and sodium tartrate and 2 grammes of 
diacid-sodium phosphate are added, with 10 grammes of com- 
pressed yeast, and the mixture is kept at a temperature of from 
30° to 34° C. If but little sugar is present, two to three hours 
will be sufficient. 

That portion of t]je urine of which the specific gravity is deter- 
mined before fermentation should really be treated in the same man- 
ner. It will suffice, however, to add 0.022 to the specific gravity 
found, to make up for the increase that would otherwise be observed 
in the second specimen owing to addition of the salts. 

In every case the urine must be perfectly fresh, as fermenta- 
tion generally begins spontaneously, even after standing a short 
time. 

Einhorn's Method. — This will answer very well for ordinary 
purposes. Two especially constructed and graduated saccharimetric 
tubes (Fig. 113) are used, one of which is filled with a mixture of 
the suspected urine and yeast, and the other Avith normal urine and 

1 Roberts, Lancet, 1862, i. p. 21. Worm-Muller, Piluger's Archiv, 1884, vol. xxxiii. 
p. 211, and 1885, vol. xxxvii. p. 479. 



CA RB OHYDRA TES. 



527 



Fig. 114. 




yeast, as a control. The tubes are set aside at a temperature of 

from 30° to 34° C, when the percentage- amount of sugar in the 

urine is read off from the column of carbon 

dioxide formed. Should the second tube 

also show a small amount of gas, the figure 

corresponding to this amount is deducted 

from the first. 

Lohnstein's Method. — A very conve- 
nient modification of Einhorn's instrument, 
and one furnishing more accurate results, 
has been introduced by Lohnstein. 1 As 
will be seen from the accompanying figure 
(Fig. 115), this is essentially a U-tube open 
at both ends. The longer limb is closed 
during the process of fermentation by a 
ground-glass stopper. This stopper is pro- 
vided with an air-hole, to which a similar 
hole corresponds in the drawn-out portion 
of the tube. The apparatus is filled with the Flask for the approximate 
urine to be examined, through the bulb A, ££S& °(v. j^ksch^ fer * 
while the two air-holes at B are in commu- 
nication. Care should be had that the liquid stands exactly at the 
mark 0. The stopper is then turned so that all communication 
between the air and the urine is cut off. A little mercury is finally 
poured into the saccharimeter, when the instrument is placed in a 
vessel containing water at 35°-40° C, and maintained at a temper- 
ature of about 30° C. After twelve hours the percentage of sugar 
is read off directly. 

Precautions : 1. As every urine contains traces of free carbon 
dioxide, it is well to remove this by boiling if we have reason to 
suppose that only a small amount of sugar is present. Before 
adding the yeast the urine is, of course, cooled to the surrounding 
temperature. 

2. As the instrument yields satisfactory results only if the urine 
contains less than 1 per cent, of sugar, it is necessary to dilute it 
with water when more is present. The specific gravity may here 
serve as an index; urines of a specific gravity up to 1.018 
are examined directly; from 1.018 to 1.022 they are diluted 
twice, from 1.022 to 1.028 five times, and those above 1.028 ten 
times. 

3. A test-tube, provided with the necessary marks to indicate the 
degree of dilution of the urine, accompanies the instrument. In 
every case a globule of yeast, approximately 6-8 mm. in diameter, 



1 T. Lohnstein, " Ein neues Gabrungssaccharometer," Berlin, klin, Woch., 



528 



THE URINE. 



Fig. 115. 



is added to the urine and shaken in the tube until an even suspen- 
sion has been reached. 1 

Polammetmc Method. — For this purpose the saccharimeter of 
Soleil-Ventzke is very convenient (Fig. 116). This consists essen- 
tially of a Mcol prism, A, which may be rotated about the axis of 
the apparatus ; a second Nicol prism, at D ; vertically placed com- 
pensating prisms, consisting of dextrorotatory quartz, at E, which 

may be moved horizontally by means of 
a rack-and-pinion adjustment, turned by 
a milled head at K, so that light can pass 
through a thicker or thinner layer of the 
dextrorotatory quartz. At F is a plate 
of lsevorotatory quartz cut perpendicularly 
to the optical axis, and covering the en- 
tire field of vision ; at H biquartz plates 
of Soleil, and at I an Iceland-spar crystal ; 
BC represents a small telescope, by means 
of which the biquartz plates can be accu- 
rately focussed. When the compensation- 
prisms of this apparatus are in a certain 
position the lsevorotation of the plate F 
will be exactly compensated, and the two 
halves of the field of vision present the 
same color, while the zero of the scale X 
will coincide with the zero of the vernier 
Y, arranged on the upper surface of the 
compensators. Any change in this posi- 
tion produced by turning the screw K 
will cause the appearance of a different 
color in each half of the field of vision. 
If now, with a zero-position, an optically 
active dextrorotatory or laevorotatory sub- 
stance is interposed, the color of each half of the field of vision will 
become altered, but may be equalized again by changing the position 
of the compensators, the degree of change necessary to produce this 
result constituting an index of the power of rotation of the solution 
interposed in the tube M. 

Soleil- Ventzke's apparatus is constructed in such a manner that 
if a solution of glucose is employed, the length of the tube M being 
10 cm., every entire line of division on the scale will indicate 1 per 
cent, of sugar. 

The tube of the saccharimeter should be carefully washed out with 
distilled water, and at least once or twice with the filtered urine, 
when it is placed on end upon a flat surface and filled with the 

1 Lohnstein's saccharimeter may be procured from R. Kallmeyer & Co., Oanrein- 
burger Str. 45, Berlin. 




Lohnstein's saccharimeter. 



CARBOHYDRATES. 



529 



urine, so that this forms a convex cup at the end. The glass 
plate is now carefully adjusted, so as to guard against the admission 
of bubbles of air. The metallic cap is placed in position, care 
being taken to avoid undue pressure. The examinations are made 
in a dark room ; an ordinary lamp is used, and several readings are 
taken, until the differences do not amount to more than 0.1 or 0.2 
per cent. The tubes should be thoroughly cleansed immediately after 
the experiment. 

' In every case the filtered urine should be free from albumin, and, 
if markedly colored, should be previously treated with neutral lead 
acetate in substance and filtered. 

If it is desired to demonstrate only the presence of sugar, the 
compensators are first brought to the zero-position. If now, upon 

Fig. 116. 




Soleil-Ventzke's saccharimeter. 



interposition of the tube filled with urine, a difference in the color 
of the two halves of the field of vision is noted, the presence of an 
optically active substance in the urine may be assumed ; and if the 
deviation is at the same time to the right, the presence of glucose is 
rendered highly probable, while a deviation to the left will generally 
be referable to levulose or /?-oxybutyric acid. Indican, peptones 
(albumoses), cholesterin, and certain alkaloids, it is true, also turn 
the plane of polarization to the left ; but as a rule these substances 
need not be considered, as cholesterin occurs but rarely, and indican 
is usually present in only small amounts in diabetic urines. Albu- 
moses, if present, must first be removed. Lactose and maltose, 
which also turn the plane of polarization to the right, may be dis- 

34 



530 THE URINE. 

tinguished from each other and from glucose by the phenylhydrazin 
test. Levulose turns the plane of polarization to the left. Oxy- 
butyric acid is practically always associated with the presence of 
glucose, and may be recognized by allowing the urine to undergo 
fermentation, when the filtered urine will become distinctly lasvo- 
rotatory. 

Bremer's Diabetic Urine Test. 1 — The test is based upon the 
different behavior toward certain anilin dyes of diabetic, as compared 
with non-diabetic, urine. If a trace of a mixture of 2 parts of eosin 
and 3 parts of gentian- violet, for example, is added to non-diabetic 
urine, it will be observed that the urine gradually dissolves the eosin 
and assumes a yellowish or bright-red color, while the gentian- violet 
fails to dissolve. If diabetic urine, on the other hand, is treated in 
the same manner, the eosin will likewise dissolve, but a solution of 
the gentian-violet also occurs, and the entire specimen eventually 
assumes a violet color. 

Of late, Bremer has advised the use of Merck's gentian-violet B, 
or of methyl-violet 5B. The test is extremely simple : two well-dried 
test-tubes are filled to about one-half, the one with normal urine and 
the other with the urine to be examined. About 0.5 mgrm. of either 
of the above reagents is then placed upon the surface of the urine ; 
the tubes are kept in a warm place or immersed in warm water. 
On standing, streaks of blue gradually appear in both specimens, 
but on shaking the color disappears in the normal specimen, while 
the entire bulk of the diabetic urine assumes a blue or violet color. 
A reddish-purplish color is often observed in non-diabetic specimens, 
but is of no significance. Bremer admits that doubtful results may 
be obtained with urines presenting a specific gravity below 1.014 or 
1.015, and that in such cases it may be impossible to distinguish 
non-diabetic from diabetic urine. He claims, on the other hand, 
that a positive result with a urine of high specific gravity is pathog- 
nomonic of diabetes, and that this may be obtained even at a time 
when the sugar has temporarily disappeared from the urine. 

The substance which gives rise to this peculiar reaction is un- 
known. Sugar in itself, as also acetone and diacetic acid, are not 
concerned in its production. The reaction of the urine also is unim- 
portant. Bremer is inclined to believe that in non-diabetic urines 
one of the coloring principles helps to render the urine refractory. 
As he says, colorless diabetic urines yield the most striking color- 
reactions, and especially those in which a greenish shimmer is 
apparent. 

On the whole, Bremer's observations have been confirmed so far 

1 L. Bremer, " Anilinfarbenproben d. Harris bei Diabetes," Centralbl. f. inn. Med., 
vol. six. p. 307. T. B. Futcher, Pbila. Med. Jour., 1898. L. Bremer, " On the Chemi- 
cal Behavior of Eosin and Gentian-violet toward Normal and Diabetic Urines," N. Y. 
Med. Jour., 1897. 



CARBOHYDRATES. 531 

as diabetic urine is concerned. Exceptions, however, occasionally 
occur even in cases of true diabetes, and, as Bremer admits, positive 
results are frequently observed in urines of a low specific gravity. 

The test is of interest, and may possibly be further modified so as 
to be of value in diagnosis, but as yet it would scarcely be warrant- 
able to draw definite conclusions from its occurrence, even when the 
specific gravity is high. 

Lactose. — Lactose may be found in the urine toward the end of 
gestation, but it occurs more especially in nursing-women in whom 
the flow of milk is impeded. It is generally stated, however, that 
•lactosuria also occurs in nursing-women who have well-developed 
breasts, in the absence of any obstruction, and that the good qual- 
ities of a wet-nurse are indicated by a copious and persistent elim- 
ination of milk-sugar. Its presence may be inferred if a positive 
result is obtained with Trommer's and Nylander's tests, while the 
phenylhydrazin and fermentation tests give negative results, although 
an osazon can be obtained from the isolated substance, and although 
lactose undergoes a certain form of alcoholic fermentation. 

Lemaire, who has recently investigated this subject, found that 
the urine of nineteen women examined in this direction apparently 
contained no sugar during the last twelve days preceding confine- 
ment (Trommer's and Nylander's tests), while a positive reaction was 
obtained with Trommer's reagent in two cases and with Nylander's 
reagent in thirteen cases after confinement. The phenylhydrazin 
test was negative in all nineteen before and positive after confine- 
ment, when this was directly applied to the substance isolated according 
to Baumann's method. The percentage varied between 0.013 and 
0.438, and appeared to be uninfluenced by the act of nursing. 1 

Lsevulose. 2 — It is claimed that lsevulose is occasionally found in 
diabetic urines together with glucose, and may also occur spontane- 
ously unaccompanied by glucosuria. Such urines show a deviation 
to the left or none at all, while the other tests for sugar indicate the 
presence of a reducing substance. 

Maltose. — Maltose, together with glucose, was first found in the 
urine of a patient supposedly the subject of paucreatic disease, asso- 
ciated with an acholic condition of the stools. Since that time it 
has been repeatedly observed in diabetic patients. In one case the 
amount was 27.8 grammes pro liter. Similar results have been ob- 
tained in dogs after extirpation of the pancreas. 3 Its recognition 
is practically dependent upon the formation of its osazon and a deter- 
mination of the melting-point of the latter. Such urines, moreover, 

1 De Sinety, Maly's Jakresber., 1874, vol. iii. p. 134. Hempel, Arch. f. Gynaek., 
1875, vol. viii. p. 312. Ney, Ibid., 1889, vol. xxxv. p. 239. F, Hofmeister, "Ueber 
Laktosurie," Zeit. f. physiol. Chem., 1877, vol. i. p. 101 (lit.). F. A. Lemaire, Ibid., 
1896, vol. xxi. p. 442. 

2 Seegen, Centralbl. f. d. med. Wiss., 1884, vol. xxii. p. 753. H. Eosin and L. La- 
baud, Zeit, f. klin. Med., vol. xlvii. Hefte 1 u. 2. 

3 Lepine and Boulud, Compt. rend., vol. cxxxii. p. 610. 



532 THE URINE. 

show a larger percentage of sugar on polarization t T nan on titration 
with Fehling's solution. At the same time it will be observed that 
on heating for two hours with hydrochloric acid at 106° F. the 
polarimetric values become smaller, while the titration values 
increase. 

Dextrin. 1 — In one case of diabetes dextrin appeared to take the 
place of glucose. It may be recognized by the fact that upon the 
application of Fehling's test the blue liquid first becomes green, 
then yellow, and sometimes dark brown. Traces of dextrin are 
probably present in every urine, but cannot be demonstrated with 
the common tests. 

Laiose. 2 — Laiose has been found in the urine of a diabetic patient. 
It is essentially characterized by the fact that on titration with 
Fehling's solution from 1.2 to 1.8 per cent, more sugar is indicated 
than by the polarimetric method. 

Pentoses. — Traces of pentoses, viz., xylose, arabinose, and rham- 
nose, may be found in every urine. Larger quantities were first 
observed by Salkowski and Jastrowitz, in the urine of a morphine 
habitue, in which the pentosuria alternated with glucosuria. A sim- 
ilar case was reported by Real. Kiilz and Vogel found larger quan- 
tities in a case of diabetes ; and still more recently Bial has reported 
two instances which occurred in apparently healthy individuals. A 
digestive pentosuria has also been described. Such urines reduce 
Fehling's solution and Nylander's solution, and give rise to the for- 
mation of an osazon when treated with phenylhydrazin. The osazon, 
how T ever, can be readily distinguished from that obtained from glu- 
cose, maltose, or lactose, etc., by the melting-point (159°-160° C). 
The fermentation test is negative. Xylose and rhamnose turn the 
plane of polarization to the right, while arabinose is optically inac- 
tive. The presence of pentoses can be definitely established with 
Tollens' orcin test : 

Tollens' Orcin Test. — A few granules of orcin are dissolved in 
4 to 5 c.c. of concentrated hydrochloric acid by the aid of heat, 
so that a slight excess is present. This solution is divided into 
two equal parts and allowed to cool. To one portion 0.5 c.c. 
of the urine to be examined is added, and to the other an equal 
amount of normal urine of the same specific gravity. Both speci- 
mens are placed in a beaker containing boiling water, when in the 
presence of pentoses a green color will first be observed at the top, 
which gradually extends throughout the mixture, while the normal 
specimen scarcely changes in color. In the presence of 0.1 per 
cent, a positive reaction is still obtained, which is especially marked 
if the urine has been previously decolorized with animal charcoal. 
The green pigment which results can be extracted by shaking with 

1 Reichard, Maly's Jahresber., 1876, vol. v. p. 60, 

2 Leo, Virchow's Arcbiv, vol, cvii, 



GLUCURONIC ACID. 533 

amyl alcohol, and on spectroscopic examination it gives rise to a 
well-defined band of absorption in the red portion of the spectrum 
near the yellow border. 

Tollens' phloroglucin test, in which phloroglucin is substituted for 
the orcin, and in which a deep-red color is obtained in the presence 
of a pentose, may also be used, but the reagent indicates the presence 
of glucuronates as well. 

Very curiously, the pentosuria persists even though no carbo- 
hydrates are ingested ; and there is evidence to show that pentoses 
are formed within the body. As a matter of fact, Hammarsten has 
succeeded in demonstrating the presence of a pentose among the 
decomposition-products of a nucleo-glucoproteid which is found in 
the pancreas ; and Blumenthal arrived at similar results in the case 
of various nucleinic acids which occur in the animal body. It is 
possible, on the other hand, that the pentoses may result from the 
metabolic products of glucose which are formed under normal con- 
ditions by a process of oxidation, and are then eliminated as such 
under still unknown influences. 

Aside from the traces normally present in the urine, pentosuria 
must be regarded as a metabolic anomaly, analogous to glucosuria, 
cystinuria, alkaptonuria, etc. 

Literature. — E. Salkowski u. M. Jastrowitz, " Ueber eine bisher nicbt beobacbtete 
Zuckerart im Harn," Centralbl. f. d. med. Wiss., 1892, No. 19. E. Salkowski, " Ueber 
d. Pentosurie," Berlin, klin. Woch., 1895, No. 17. F. Blumenthal, Ibid., No. 26 ; and 
Zeit. f. klin. Med., vol. xxxvii. p. 415. E. Kulz u. J. Vogel, Zeit. f. Biol., N. F., 1896, 
vol. xiv. p. 189. E. Salkowski, " Ueber d. Vorkommen von Pentosen im Harn," Zeit. 
f. physiol. Chem., 1899, vol. xxvii. p. 587. Bial, Ueber Pentosurie, Zeit. f. klin. Med., 
1900, vol. xxxix. p. 472. 

Animal Gum. — Landwehr's animal gum, according to modern 
researches, is a constant constituent of normal urine, but is of no 
clinical interest. Of the chemical nature of the substance not much 
is known, but there is evidence to show that in all probability the 
body is a derivative of chondroitin-sulphuric acid. 

GLUCURONIC ACID. 

Glucuronic acid is derived from glucose, and constitutes an inter- 
mediary product of the normal metabolism of the body. In the 
urine it is found only in combination with certain fatty and aromatic 
alcohols, forming compounds which are related to the glucosides 
and are generally spoken of as the conjugate glucuronates. Such 
bodies have been observed in the urine following the ingestion of 
chloral, camphor, naphtol, oil of turpentine, menthol, phenol, mor- 
phin, antipyrin, etc., and traces may also be obtained from nor- 
mal urines. The normal glucuronates are undoubtedly compounds 
of glucuronic acid with phenol, paracresol, indoxyl, and skatoxyl. 



534 THE URINE. 

Their amount is exceedingly small, as the greater portion of 
these bodies is normally eliminated in combination with sulphuric 
acid. 

Of the quantitative variations of the normal glucuronates and 
their relation to disease, next to nothing is known. Their clinical 
interest centres in the fact that certain glucuronates are capable of 
reducing copper and bismuth in alkaline solution, and may thus be 
confounded with glucose. Such urines, however, do not undergo 
fermentation. The glucuronates turn the plane of polarization to the 
left, while glucuronic acid itself is dextrorotatory. Like the pen- 
toses, the glucuronates give a positive reaction with phloroglucin, 
while they do not react with orcin (see page 532). With the free 
acid phenylhydrazin forms crystalline compounds (see page 520). 

Literature. — H. Thierfelder, "Ueber d. Bildung v. Glykuronsaure," etc., Zeit. 
f. physiol. Chem., 1886, vol. x. p. 163 ; "Untersuchungen iiber d. Glykuronsaure," Ibid., 
1887, vol. xi. p. 388. P.Mayer, "Ueber d. Ausscbeidung u. d. Nacbweis d. Glyku- 
ronsaure," Berlin, klin. Woch., 1899, pp. 591 and 617. P. Mayer u.C. Neuberg, Zeit. f. 
pbysiol. Cbem., 1900, vol. xxix. p. 256. 

INOSIT. 

According to Hoppe-Seyler, traces of inosit may be found in 
the urine under normal conditions. Somewhat larger quantities 
are eliminated following the ingestion of large amounts of water, 
and for this reason possibly inosituria is notably observed in cases 
of diabetes insipidus, in diabetes mellitus, and in chronic intersti- 
tial nephritis. Its occurrence in these diseases is, however, not 
constant. The substance is devoid of clinical interest. It is not a 
carbohydrate, but belongs to the aromatic series, and is commonly 
regarded as hexa-hydroxybenzol. Its formula is C 6 H 12 6 -j- H 2 0. 
For methods of isolating the substance from the urine, the reader is 
referred to special works. 1 

URINARY PIGMENTS AND CHROMOGENS. 

Under normal conditions urochrome and uroerythrin, to which 
latter the red color of urate sediments is due, are the only known 
pigments which occur preformed in the urine, while indigo-red and 
indigo-blue, derived from indoxyl sulphate and indoxyl glucuronate, 
may be artificially produced. In disease, on the other hand, various 
other pigments may be found, which occur in the urine either free or 
in the form of chromogens. Among the former may be mentioned 
haemoglobin, methsemoglobin, hsematin, hsematoporphyrin, urorubro- 
hsematin, urofuscohsematin, urobilin,the biliary pigments, and melanin; 
while abnormal chromogens are met with following the ingestion of 
certain drugs, such as santonin, senna, rheum, iodine, etc., as also in 
cases of poisoning with carbolic acid, creosote, etc. The occurrence 

1 C. E. Simon, Physiological Chemistry, Lea Bros. & Co. 



URINARY PIGMENTS AND CHROMOGENS. 535 

of some of these substances, such as the various forms of blood-pig- 
ment, the biliary pigments, and indigo, viz., indican, is of considerable 
clinical interest, while others again are of only minor importance. 

Normal Pigments. — Urochrome. — To the presence of this pig- 
ment, which appears to be identical with the normal urobilin of 
MaeMunn, but which should not be confounded with the pathological 
urobilin of Jaffe, the normal yellow color of the urine is probably 
largely due. It is supposedly derived from bilirubin, which in turn is 
referable to hsematin, and thus to the haemoglobin of the blood. From 
the bilirubin secreted into the intestinal tract it is derived by a process 
of oxidation, and not of reduction, as is generally stated (Gautier). 
Such a transformation, according to our present knowledge, may, 
however, also occur directly, without the intervention of bilirubin, 
as urochrome is found in the urine of dogs in which the bile is 
prevented from entering the intestinal tract by the establishment of 
a biliary fistula. An increased amount is similarly found in cases 
in which resorption of large extravasations of blood is taking 
place — in short, whenever an increased destruction of red corpuscles 
occurs. Under the opposite circumstances — i. e., in conditions 
associated with a new formation of red corpuscles, as in certain 
forms of anaemia, chronic parenchymatous nephritis, diabetes, dis- 
eases of the bone-marrow, etc. — it occurs in diminished amount. 
Urochrome, moreover, is present in urobilin-free feces, and even in 
those of infants with congenital atresia of the biliary ducts. 

In order to obtain urochrome from normal urine, this is acidulated 
with 1—2 grammes of dilute sulphuric acid pro liter, filtered, and 
saturated with ammonium sulphate in substance, when the flakes 
which are formed, if an excess of the salt has been added, are dried 
and treated with warm, slightly ammoniacal absolute alcohol ; the 
pigment is then obtained upon evaporation of the alcohol. 

An alcoholic solution of urochrome, like the urobilin of Jaffe, 
is said to exhibit a beautiful greenish fluorescence when treated with 
ammonia and a few drops of a solution of zinc chloride ; but, 
unlike the latter substance, its acidulated alcoholic solutions present 
a broad band of absorption at F, which extends more to the left 
than to the right of this line, while the remainder of the spectrum 
is at the same time absorbed to the right end, from a point some- 
what to the left of G. Garrod, on the other hand, states that by 
acting upon urochrome with acids he did not succeed in obtaining 
any product showing the urobilin band or yielding the well-known 
fluorescence with zinc chloride and ammonia. But a substance 
having both these properties was readily obtained by the action of 
aldehyde upon an alcoholic solution of the pigment. In a short 
time — shorter still when the liquid is warmed — an absorption-band 
appears like that of urobilin, and the tint of the solution deepens 
to a rich orange-yellow. With zinc chloride and ammonia a bril- 
liant green fluorescence appears, and the band is shifted toward the 



536 THE URINE. 

red, as that of urobilin is under like circumstances. The process 
can be stopped at this point by the simple addition of water, for 
aldehyde has no such action upon aqueous solutions of urochrome. 
If, however, the action be allowed to continue, a further change 
ensues ; the liquid reddens, and a second band appears in the violet. 
The fluorescence can still be obtained with zinc chloride and am- 
monia, and both bands are shifted toward the red and are closer 
together than before. The reaction with aldehyde, according to 
Garrod, affords a very delicate test for the presence of urochrome 
in alcoholic solutions. The product of the earlier stage, although 
it is not identical with urobilin, resembles that pigment quite as 
closely as the products obtained from bilirubin and hsematin by the 
action of reducing agents ; but no second band is developed when 
aldehyde is added to an alcoholic solution of urobilin. 1 

By the action of potassium permanganate upon urobilin Biva 
and Chiodera 2 obtained a substance closely resembling urochrome, 
and a similar product is formed when an aqueous solution of uro- 
bilin containing ether is evaporated upon a water-bath. Neither 
product shows any absorption-band, and both behave as urochrome 
does when it is acted upon by aldehyde. 

Uroerythrin. — Uroerythrin is the pigment which imparts the red 
color to crystals of uric acid and the pink tint to urate sediments. 
Under strictly normal conditions it probably does not occur in the 
urine, but it readily appears with the slightest deviation from 
health, and when present in larger amounts imparts a deep-orange 
color to the urine. Under pathological conditions it is seen espe- 
cially in cases of hepatic insufficiency, in which the liver, owing 
to a greatly increased destruction of red corpuscles, is unable to 
transform into bile-pigment all the blood-pigment which is carried 
to it. It also occurs when an absolute insufficiency on the part of 
the hepatic cells exists, so that the organ is not even capable of 
causing the transformation of a normal amount of haemoglobin. 
Uroerythrin is thus seen in notable quantities in cases of cirrhosis 
and carcinoma of the liver, in passive congestion resulting from 
heart-disease, in acute articular rheumatism, gout, pneumonia, 
malarial fever, erysipelas, spinal curvature, etc. In typhoid fever 
a marked excretion of uroerythrin is exceptional, and its occurrence 
has been associated with pulmonary complications. In nephritis 
it is seldom found in the urine, but Garrod cites an instance of 
pneumonia in which an abundant excretion of the substance accom- 
panied conspicuous albuminuria. 

In certain diseases, such as hepatic cirrhosis, the excretion of 
uroerythrin, as also of urobilin, is said to be much diminished 
when the patient is placed upon a milk-diet (Biva). 

1 A. E. Garrod, " The Bradshaw Lecture on the Urinary Pigments in Their Patho- 
logical Aspects," Lancet, Nov. 10, 1900. v 
^2Eiva and Chiodera, Arch. ital. di Clm. Med., 1896, vol. xxxv. p. 505. 



URINARY PIGMENTS AND CHROMOGENS. 537 

Chemically, its relation to haemoglobin, hsematoidin, and bilirubin 
is seen from the following analyses of the various pigments : 

C H N O S Fe 

Haemoglobin, 53.85 7.32 16.17 . . . 0.39 0.43 

Hsematoidin, 65.05 6.37 9.51 

Bilirubin, 67.83 6.29 9.79 16.79 

Uroerythrin, 62.51 5.79 3L70 

When present in large amounts uroerythrin is readily recognized 
by the salmon-red color which it imparts to urinary sediments. 
Otherwise it is best to precipitate the urine with neutral lead acetate, 
barium chloride, or a similar reagent, when in the absence of uro- 
erythrin a milky -white precipitate is obtained, while a pale rose- 
colored sediment indicates the presence of the pigment in appreciable 
amounts; a more pronounced rose color is produced if large quan- 
tities are present. In every case at least ten to fifteen minutes 
should be allowed to elapse before forming a definite conclusion, so 
that the sediment may have abundant time to settle. 

The pigment itself is unstable. Its solutions in alcohol or 
chloroform are rapidly decolorized by light, and even when kept in 
the dark quickly undergo change. Alkalies destroy the pigment 
readily, with the production of a green tint. Neutralization of the 
alkali does not restore the original color or bring back the absorption 
spectrum, which is characteristic, though ill-defined, consisting of 
two faint bands in green and blue, united by a fainter shading. 
One of these bands has the position of the urobilin band, but both 
alike disappear when the solutions are decolorized by light. The 
pigment is readily soluble in amyl alcohol and acetic ether (Garrod). 1 

Normal Chromogens. — The chromogens occurring in normal 
urine are indican, urohsematin, and an unknown chromogen which 
yields urorosei'n when treated with mineral acids. 

Indican. — It has been pointed out (see Sulphates) that the indol 
formed during intestinal putrefaction is oxidized to indoxyl in the 
blood ; this, entering into combination with sulphuric acid, is elimi- 
nated in the urine as sodium or potassium indoxyl sulphate, or 
indican, as represented by the equations : 



(1) G 8 H 7 N 


+ o 


= C 8 H 7 NO 


Indol. 




Indoxyl. 


(2) C 8 H 7 NO 


/OH 
X OH 


/C 8 H 6 NO 
= S0 2 < +H 2 
x OH 


Indoxyl. 




Indoxyl suiphate. 


.C 8 H 6 NO 
(3) S0 2 < + Na 2 HPO, 
x OH 

Indoxyl sulphate. 


/C 8 H 6 NO 
= S0 2 < + NaH 
' X)Na 

Indoxyl-sodium 
sulphate. 



1 A. E. Garrod, loc. cit. A. Bobin, Urologie clinique de la Fievre typhoide, Paris, 1877. 



538 THE URINE. 

Formerly it was thought that indican was also formed within 
the tissues of the body in the absence of putrefactive organisms. 1 
Further researches, however, have demonstrated that micro-organ- 
isms are always concerned in the production of indican, and that 
in health the large intestine is its sole source. Baumann, who 
succeeded in absolutely disinfecting the intestinal tract of a dog by 
means of large doses of calomel, thus observed that all traces of 
indican, as also of phenol and paracresol, disappeared from the 
urine. According to Senator, moreover, indican does not occur 
in the urine of newly born infants which have not as yet 
received nourishment. This observation is a strong point in 
favor of Nencki's teachings that indol is a specific product of 
albuminous putrefaction in the presence of organized ferments, 
as putrefiable substances are here present, but no putrefactive 
organisms. Tuczek's observations on abstinence from food in 
cases of insanity, in which indican was observed in the urine only 
when albumins, though in minimal amounts, were ingested, also 
speak very strongly against Salkowski's theory. Finally, it has been 
demonstrated that in cases in which an artificial anus is established 
near the distal end of the ileum the conjugate sulphates disappear 
almost entirely from the urine, while they reappear in normal amount 
as soon as the connection between the small and large intestines has 
been re-established. 2 

The amount of indican which is normally eliminated in the urine 
varies somewhat with the character of the diet. Jaffe 8 obtained 6.6 
mgrms. from 1000 c.c. of urine, as an average of eight obser- 
vations. The largest quantities excreted in health are found after a 
liberal indulgence in animal food, particularly the so-called red 
meats, while the smallest amounts are observed during a milk- or 
kefir-diet. By means of the latter article, indeed, the greatest dimi- 
nution in the degree of intestinal putrefaction may be effected in 
man. 

In pathological conditions an increased elimination of indican is 
observed : 

1. In all diseases which are associated with an increased degree 
of intestinal putrefaction. As there appears to be little doubt that 
this is largely regulated by the acidity of the gastric juice, an in- 
creased indicanuria, according to personal observations, is encountered 
when anachlorhydria or hypochlorhydria exists. It has been pointed 
out elsewhere that it is possible to form a fairly accurate idea of the 
amount of free hydrochloric acid in the gastric juice by an examina- 

1 E. Salkowski, Ber. d. den tech. chem. Ges., 1876, vol. ix. pp. 138 and 408. Bau- 
maiin, Zeit. f. physiol. Chem., 1886, vol. x. p. 123. Senator, Centralbl. f. d. med. Wiss., 
1877, vol. xv. pp. 357, 370, and 388. 

2 Nencki, Macfadyen u. Sieber, Arcb. f. exper. Path. u. Pharmakol., 1891, vol. xxix. 

3 Jaffe, Centralbl. f. d. med. Wiss.. 1872, vol. x. pp. 2, 481, and 497; and Virchow's 
Archiv, 1877, vol. lxx. p. 72. 



URINARY PIGMENTS AND CHROMOGENS. 539 

tion of the urine in this direction. Large quantities of indican are 
thus eliminated in cases of carcinoma of the stomach, and exceeded 
only by those observed in cases of ileus, so that this symptom, in 
my estimation, is of considerable value in differential diagnosis, 
and is one, moreover, which has not received the attention it 
deserves. Exceptions to this rule are at times, though rarely, 
met with, for which it is, however, impossible to account at 
present. Large quantities of indican are also observed in cases of 
acute, subacute, and chronic gastritis. In the course of personal 
observations in this direction I was impressed with the curious 
phenomenon that in cases of ulcer of the stomach, notwith- 
standing the simultaneous occurrence of hyperchlorhydria, an 
increased elimination of indican, contrary to what is usually seen in 
hyperchlorhydria referable to other causes, is quite constantly found. 
Possibly the existence of muscular atony which was noted in those 
cases may serve to explain this apparent incongruity, but it is as yet 
impossible to offer a satisfactory explanation of the phenomenon. 
Remembering the origin of indican, and the relation which the 
amount eliminated bears to the degree of intestinal putrefaction, it 
will be unnecessary to enumerate the long list of diseases in which 
an increased indicanuria has been observed, as it will be found that 
in the majority of these cases the indicanuria is merely an index 
of the condition of the gastric juice and the motor power of the 
stomach. 1 

2. It should be noted that in cases in which the peristaltic move- 
ments of the small intestine have become impeded, as in ileus, acute 
and chronic peritonitis, an increased elimination of indican will inva- 
riably take place, no matter what the state of the gastric juice may 
be. In such conditions, and especially in ileus, the largest quanti- 
ties are observed, a point which may be of decided value in differ- 
ential diagnosis, as diseases of the large intestine alone are never 
associated with an increase in the amount of indican. In simple, 
uncomplicated constipation increased indicanuria is not seen; and 
should an examination in such cases reveal the presence of more 
indican than normal, it will be safe to assume the existence of disease 
elsewhere, and especially of the stomach. 

3. As albuminous putrefaction may also take place within the 
body, an increased indicanuria is observed in cases of empyema, 
putrid bronchitis, gangrene of the lung, etc. ; but while in the con- 
ditions mentioned above the indol-producing organisms appear to be 
especially active, the elimination of phenol in the latter condition 
may be more pronounced at times than that of indican. Bearing in 
mind the points here set forth, I cannot agree with others in saying 
that the study of indicanuria possesses no importance from a clini- 

1 C. E. Simon, " Indicanuria," Am. Jour. Med. Sci. (full literature), 1895, vol. ex. 
p. 48. 



540 THE VRINE. 

cal standpoint. I maintain, on the other hand, that an examina- 
tion of the urine in this direction is at least as important as the testing 
for albumin and sugar, and that points of decided importance, not 
only in diagnosis, but cdso in prognosis and treatment, may thus be 
gained. 

Of interest in this connection also is the observation that in cases 
of increased indicanuria oxalate sediments are not uncommonly ob- 
served ; but I am not willing to admit, as Harnack and van der 
Ley en suggest, that the indicanuria which follows the ingestion of 
small doses of oxalic acid is produced by a toxic action of the acid 
upon the tissue albumins. In these cases also the increased indican- 
uria is referable to increased intestinal putrefaction. 1 

When indican is treated with hydrochloric acid, it is decomposed 
into sulphuric acid and indoxyl ; should an oxidizing substance be 
present at the same time, indigo-blue, the blue coloring-matter of 
the urine, results : 

2C 8 H 6 NKS0 4 + 20 = C 16 H 10 N 2 O 2 + 2HKS0 4 . 
Potassium, indoxyl Indigo-blue, 

sulphate. 

Indigo-blue in small amounts may be found free in the sediment 
of almost every decomposing urine, usually occurring in the form of 
small, amorphous granules, and more rarely in crystalline form. 
Urines have, however, also been observed which were blue when 
passed, or which turned blue as a whole upon standing. Such a 
phenomenon must be regarded as a medical curiosity. Undoubtedly 
it is referable to the action of micro-organisms (see page 581), 
although McPhedran and Goldie mention that in their case bacteria 
were present only in small numbers. 2 

The blue pigment which may be obtained from urines has been 
variously described as Prussian-blue, urocyanin, cyanurin, Harn- 
blau, uroglaucin, choleraic urocyanin, but it has been shown 
to be indigo-blue, and derived from a colorless mother-substance 
which is present in every urine to a greater or less extent, and 
which has been named indican. This has been shown to be identi- 
cal with the uroxanthin of Heller and Thudichum's choleraic uro- 
cyaninogen. 

Tests for Indican. — The urine of twenty-four hours is care- 
fully collected and a specimen taken for examination. A few cubic 
centimeters are then mixed with an equal volume of Obermayer's 
reagent, and shaken with a small amount of chloroform. Ober- 
mayer's reagent is a 2 pro mille solution of ferric chloride in concen- 
trated hydrochloric acid. 3 

1 v. Moraczewski, " Oxalurie and Indicanurie," Cent. f. inn. Med., 1903, No. 1. 

2 A. McPhedran and W. Goldie. "A Case of Indigosuria," Trans. Assoc. Am. Phys., 
1901, vol. xvi. p. 242. 

3 Obermayer, Wien. klin. Woch., 1890, vol. iii. p. 176. 



URINARY PIGMENTS AND CHROMOGENS. 541 

Stokvis' modification of Jaffe's test may also be employed. 1 To 
this end, a few cubic centimeters of urine are treated with an equal 
volume of concentrated hydrochloric acid, and two or three drops 
of a strong solution of sodium or calcium hypochlorite. The mixt- 
ure is shaken with 1 or 2 c.c. of chloroform as above. The indigo 
which is set free in this manner is taken up by the chloroform, and 
colors this blue to a greater or less extent, the degree of increase, as 
compared with the normal, being determined by the intensity of the 
color. Albumin need not be removed. Bile-pigment, which inter- 
feres with the reaction, is removed by means of a solution of lead 
subacetate, which is carefully added in order to avoid an excess. 
Urines presenting a very dark color may be cleared in the same 
manner. Potassium iodide, owing to the liberation of free iodine, 
will color the chloroform more or less of a carmine. For the sake 
of comparison, it is well to employ the same quantities of urine and 
reagents in every case, marked tubes being very convenient for this 
purpose. 

The method last described I have also found to be a fairly sensi- 
tive test for albumin, in the presence of which a well-marked cloud 
appears near the surface of the mixture and gradually extends 
downward. 

Quantitative Estimation. — Wang's Method? — The method is 
based upon the decomposition of potassium indoxyl sulphate by 
means of concentrated hydrochloric acid and the oxidation to indigo- 
blue of the indoxyl which is thus formed. The indigo-blue is fur- 
ther transformed into indigo-sulphuric acid, and this titrated with 
a solution of potassium permanganate of known strength. The 
various changes which take place are represented by the following 
equations : 



(1) C 8 H 6 NS0 4 K + 
Indican. 


H 2 


= C 8 H 6 N.OH 
Indoxyl. 


+ HKS0 4 . 


(2) 2C 8 H 6 N.OH + 
Indoxyl. 


20 


= C 16 H 10 N 2 O 2 
Indigo-blue. 


+ 2H 2 0. 


(3) 


C 16 H 10 N 2 O 2 + 2H 2 S0 4 
Indigo-blue. 


= C 16 H 8 (HS0 3 ) 2 N 2 2 + 2H 2 0. 
Indigo-sulphuric acid. 


>C 16 H 10 N 2 O 2 
Indigo-blue. 


+ 4KMn0 4 + 6H 2 S0 4 


5C, 6 H 10 N 2 O 4 + 2K 2 S0 4 + 4MnS0 4 +6H 2 0. 



Reagents required : 1 . A 20 per cent, solution of lead acetate. 

2. Obermayer's reagent. This is a 2 pro mille solution of ferric 
chloride in concentrated hydrochloric acid (sp. gr. 1.19). 

3. Chloroform. 

4. Concentrated sulphuric acid. 

1 See Senator, Centralbl. f. d. med. Wiss., 1877, vol. xv. p. 257. 

2 E. Wang, " Ueber d. quantitative Bestimmung d. Harnindikans," Zeit. f. physiol. 
Chem., vol. xxv. p. 406. 



542 THE URINE. 

5. A mixture of equal parts of alcohol (96 per cent.), ether, and 
water. 

6. A concentrated solution of potassium permanganate — i. e., a 
solution containing about 3 grammes pro liter. The titration is 
conducted with this solution diluted in the proportion of 5 c.c. to 
195 c.c. of water. Its titre is ascertained before each titration by 
comparing it with a dilute solution of oxalic acid of known strength ; 
for example, one containing 0.1 gramme of the acid dissolved in 100 
c.c. of water, as described on page 450. The amount of indigo-blue 
which each cubic centimeter will represent is ascertained by multi- 
plying the corresponding amount of oxalic acid by 1.04. 

Example. — Supposing that the permanganate solution is found of 
such strength that 1 c.c. represents 0.00014 gramme of oxalic acid; 
the corresponding amount of indigo would be 0.00014 X 1.04 = 
0.00015 gramme. 

Method. — The urine is first examined for indican, as described 
above. Should a very intense reaction be thus obtained, only 25 or 
50 c.c. are used for the quantitative estimation, while larger amounts 
are taken (200—500 c.c.) if the reaction is of only moderate intensity 
or negative altogether. 

The urine is precipitated with lead acetate solution, care being 
taken to avoid an excess. A large and accurately measured 
portion of the clear nitrate is treated in a separating funnel with an 
equal volume of Obermayer's reagent and extracted with chloroform. 
To this end, 30 c.c. are added at a time and shaken for one minute. 
Two or three extractions are usually sufficient to remove the entire 
amount of indigo. The extract is placed in a small flask, and the 
chloroform distilled off. The residue is dried for a few minutes on 
a water-bath until traces of remaining chloroform have been re- 
moved. It is then washed with the alcohol-ether- water mixture to 
remove the reddish-brown pigment which is present together with the 
indigo-blue. The latter remains undissolved. After filtering off any 
particles of indigo that may be in suspension, through a small filter, 
this is dried and repeatedly extracted with boiling chloroform. The 
chloroform extract is filtered into the original indigo flask, the 
chloroform distilled off, the residue dried as before, and while still 
warm treated with 3 or 4 c.c. of concentrated sulphuric acid. The 
entire residue should be brought into solution by careful agitation. 
After standing for twenty -four hours the contents of the flask are 
poured into 100 c.c. of cold water; the flask is rinsed and the 
washings added to the solution. This is filtered once more and 
titrated with the permanganate solution. At first the blue color of 
the solution changes but little ; later it turns greenish, and finally 
becomes yellowish or entirely colorless — not red. As a rule, the 
end-reaction is quite distinct, but the titration requires experience. 
The best results are obtained when from 10 to 15 c.c. of the dilute 



URINARY PIGMENTS AND CHROMOGENS. 543 

permanganate solution are used. The resulting amount of indigo 
contained in the measured-off quantity of the first filtrate is then 
ascertained as described above. 

Example. — Amount of urine : 1780 c.c. 

The stock solution of potassium permanganate contains 3 grammes 
to the liter ; 1 c.c. = 0.00596 gramme of oxalic acid == 0.0062 
gramme of indigo. Diluted solution (5 : 200) ; 1 c.c. = 0.0001*5 
gramme of indigo. 300 c.c. of urine were precipitated with 25 c.c. 
of the lead solution; 250 c.c. of the filtrate, corresponding to 230.7 
c.c. of urine, treated with 250 c.c. of Obermayer's reagent. Extracted 
twice with chloroform. 4.3 c.c. of the permanganate solution were 
used in the titration = 0.00065 gramme of indigo, corresponding to 
0.005 gramme in the 1780 c.c, according to the equation 

230.7 : 0.00065 : : 1780 : x ; x = 1157 = 0.005. 

230.7 

Other methods for the quantitative estimation of indican which 
have heretofore been used, with the exception of the spectroscopic 
method of Mtiller, are not only inaccurate, but, like this, too time- 
consuming and complicated to be of value to the practising physician. 
As a consequence almost all observers have based their conclusions 
upon an approximative estimation only. For practical purposes this 
is sufficient, and even Wang's method, though accurate and simple, 
will hardly find a ready entrance into the clinical laboratory, as it 
is still too time-consuming and too expensive for daily use. For 
scientific purposes, however, it may be recommended. 

Urohaematin. 1 — Urohaematin appears to be the chromogen of the 
red pigment of the urine, and is very likely closely related to in- 
doxyl. Little is known of its chemical composition or of its mode 
of formation. In all probability the red pigment which may be 
obtained from this substance is identical with other red pigments 
which have been described from time to time as occurring in the 
urine, such as that of Scherer, the urrhodin of Heller, the urorubin 
of Plosz, Schunk's indirubin, Bayer's indigo-purpurin, Giacosa's 
pigment, and also the indigo-red obtained by Rosenbach and Rosin 
by careful oxidation of the urine with nitric acid. 

Further investigations are necessary before this subject can be fully 
understood ; but bearing in mind the probable origin of urohaematin 
from indoxyl, it would possibly be best to speak of the red pigment 
as indigo-red. In accordance with the view that urohaematin is an 
indoxyl derivative, its clinical significance is similar to that of indican 
(which see). 

The presence in normal urine of urohaematin — i. e., a chromogen 
yielding a red pigment when treated with certain reagents — may be 
demonstrated by shaking urine with chloroform and decanting after 
1 G. Harley, Verhaudi. d. physik. med. Ges. z. Wurzburg, 1855, vol. v. p. 1. 



544 THE URINE. 

several days, when the addition of a drop of hydrochloric acid to the 
chloroform extract will cause the appearance of a beautiful rose color ; 
this varies in intensity according to the amount of the chromogen 
present. 

The purplish color so often obtained in the chloroform extract 
when Stokvis' modification of JafFe's indican test is employed is due 
to a mixture of indigo-blue and indigo-red. Indican, however, is 
generally present in larger amounts than urohsematin. In normal 
and, usually also, in pathological urines a red color is not obtained 
with the test mentioned. In a few isolated cases of ileus, peritonitis, 
and carcinoma of the stomach I have found more indigo-red than 
indigo-blue. 

The so-called " Reaction of Rosenbach " is a convenient test for 
indigo-red when this is present in increased amounts : the boiling 
urine is treated drop by drop with concentrated nitric acid, when in 
the presence of large amounts of indigo-red it assumes a dark Bur- 
gundy color, which sometimes takes on a bluish tinge when held 
to the light. Owing to a precipitation of the pigment the mixture at 
the same time becomes cloudy and the foam assumes a blue color. 
In well-marked cases the Burgundy color does not appear to be 
changed by the further addition of nitric acid, but will sometimes 
suddenly change from red to yellow when 10-20 drops of the acid 
have been added. This reaction Rosenbach l regarded as symptomatic 
of various forms of severe intestinal disease associated with an 
impeded resorption throughout the entire intestinal tract. Ewald 2 
likewise noted this reaction in cases of extensive disease of the small 
intestine, in carcinoma of the stomach, and in acute and chronic 
peritonitis ; but he obtained negative results in carcinoma of the colon, 
stricture of the oesophagus, chronic diarrhoea, etc. RosenbacNs 
reaction should be viewed in the same light as a highly increased elimi- 
nation of indican. I have met with the reaction in all conditions 
associated with greatly increased intestinal putrefaction, and, like 
Ewald, failed to note the reaction in a few cases of occlusion of the 
large intestine, in which an increased elimination of indican is like- 
wise never observed. 

Uroroseinogen. 3 — In addition to indican and urohsematin, still 
another chromogen, which yields a rose-red pigment when treated 
with mineral acids, appears to occur in normal urine, although in 
small amounts. Beyond the fact that the chromogen is not a conjugate 
sulphate, practically nothing is known of its chemical nature. The 
pigment, which has received the name urorose'in, or Harnrosa, 
appears to be identical with Heller's urophain. Urorosein is best 

1 Kosenbach, Berlin, klin. Woch., 1889, vol. xxvi. pp. 5, 490, and 520, and 1890, 
vol. xxvii. p. 585. 

2 Ewald, Ibid., 1889, vol. xxvi. p. 953. 

3 H. Rosin, Deutscb. med. Wocb., 1893, p. 51. 



URINARY PIGMENTS AND CHR03I0GENS. 545 

demonstrated by treating 5-10 c.c. of urine with an equal amount of 
concentrated hydrochloric acid, and 1 or 2 drops of a concentrated 
solution of sodium hypochlorite, when in the presence of much 
indican the mixture assumes a dark-greenish, blackish, or dark- 
blue color, owing to the formation of indigo. When the mixture 
is shaken with chloroform the supernatant fluid exhibits a beau- 
tiful rose color, which is due to the urorosein. This may now 
be extracted with amyl alcohol and separated from other pigments 
which are present at the same time, by shaking with sodium 
hydrate, whereby the solution is decolorized. Upon the addition 
of a drop or two of hydrochloric acid to the alcoholic extract the 
rose color reappears. Such solutions, however, soon become decol- 
orized upon standing. A rose-red ring, referable to this pigment, 
is also frequently obtained in pathological urines when the ordi- 
nary nitric acid test is employed. 

While normally urorosein is obtained only in traces, appreciable 
amounts are often met with in pathological conditions associated 
with grave disturbances of nutrition, as in nephritis, diabetes, 
carcinoma, dilatation of the stomach, pernicious ansemia, typhoid 
fever, phthisis, and at times in profound chlorosis, etc. A vege- 
table diet also appears to cause an increase in the amount of the 
chromogen. 

Pathological Pigments and Chromogens. — The Blood-pigments. 
— The blood-pigments proper which may occur in the urine have 
already been considered (see page 490), and in this connection it 
will only be necessary to refer briefly to the occasional presence of 
hsematin, urorubrohsematin, urofuscohsematin, and hsematopor- 
phyrin. 

H^matin is only rarely found. In order to demonstrate its pres- 
ence, the urine is rendered strongly alkaline with ammonia, filtered, 
and the filtrate examined spectroscopically, when the spectrum shown 
in Fig. 6 will be noted; this may be changed into the spectrum 
represented in Fig. 7 by the addition of ammonium sulphide. 

Urorubrohsematin and urofuscoh^ematin were observed by 
Baumstark x in the urine of a case of pemphigus leprosus compli- 
cated with visceral lepra ; they appear to be closely related to 
hsematin. The color of the urine in this case varied between 
dark red and brownish red, strongly suggesting the presence of 
blood. In order to separate the pigments, the urine was dialyzed 
and the contents of the dialyzer dissolved in sodium hydrate solu- 
tion. Upon the addition of hydrochloric acid to this solution a 
brown pigment separated out in flakes, while a second pigment 
remained in solution, imparting to it a beautiful red color. Upon 
filtration the acid filtrate was again subjected to dialysis, when the 

F. Baumstark, Pfliiger's Archiv, 1874, vol. ix. p. 568. See, also, J. W. Schultz, 
Diss., G-reifswald, 1874. 

35 



546 THE URINE. 

red pigment likewise separated out. The former substance Baum- 
stark termed urorubrohaematin, and the latter urofuscohaematin. 

Uroh^ematoporphyrin has the formula C 16 H 18 N 2 3 , and is 
probably identical with the haeniatoporphyrin resulting from the 
action of sulphuric acid upon hsematin. McMunn found a pigment 
answering the description of this substance in the urine in cases 
of rheumatism, Addison's disease, pericarditis, and paroxysmal 
hemoglobinuria, which he termed urohsematin, but which in all 
probability was hsematoporphyrin. Le Nobel found the same 
pigment in two cases of hepatic cirrhosis and in one case of crou- 
pous pneumonia. Others have likewise met with haematoporphy- 
rinuria in various forms of hepatic disease, as also in phthisis, 
exophthalmic goitre, typhoid fever, and hydroa aestivalis ; further, 
in association with intestinal hemorrhages, in cases of lead poisoning, 
and especially during long-continued use of sulphonal, trional, and 
tetronal. Nebelthau records the history of a female patient, the 
subject of congenital syphilis, who had passed dark -red urine as long 
as she could remember, and continued to do so while under observa- 
tion. Recent researches, moreover, have shown that in traces at least 
the substance is present in every urine. As regards the origin of 
these normal traces, the evidence is in favor of the view that they 
are formed within the body during its normal metabolism, and 
most likely in the liver, whence the substance is eliminated in the 
bile. A portion then escapes with the feces, while a similarly 
small amount is resorbed and eliminated in the urine. Increased 
amounts would accordingly suggest the existence of a hepatic 
insufficiency ; and, as a matter of fact, we find that actual anatom- 
ical lesions then not infrequently occur. Taylor and Sailer thus 
report that in their case of sulphonal poisoning widespread degener- 
ation of the hepatic cells existed ; and Neubauer was able to isolate 
the pigment from the liver of rabbits to which sulphonal had been 
administered, while it was absent in all other organs. On the other 
hand, it is difficult to ascribe all the phenomena of such hsemato- 
porphyrinuria to hepatic changes, seeing that changes of like degree 
may occur without conspicuous urinary abnormality, and there is 
still much that is obscure in this condition. 

Stokvis attributed the increased elimination of hsematoporphyrin 
in cases of lead poisoning and following the continued use of 
sulphonal to the occurrence of hemorrhages into the intestinal 
mucosa, and suggested that the transformation of the haemoglobin 
into hsematoporphyrin was favored by the sulphonal. But while 
intestinal hemorrhages may occur in the sulphonal cases, they are 
not always observed, and, as Garrod points out, Kast and Weiss, as 
also Neubauer, were unable to verify the recorded experiments of 
Stokvis, in which he claims to have obtained a small amount of 
hsematoporphyrin when fresh blood was digested with pepsin-hydro- 
chloric acid and sulphonal at from 38° to 40° C. 



URINARY PIGMENTS AND CHR03I0GENS. 547 

Urines which contain much hsematoporphyrin are usually dark 
red in color, but the shade may vary from a sherry or port-wine 
tint to a dark Bordeaux. It is noteworthy, however, that this color 
is not primarily due to the exaggerated degree of hsematoporphy- 
rinuria, but, as Hammarsten first pointed out, to other abnormal 
pigments which are but little known, but which are probably closely 
related to hsematoporphyrin. As Garrod says, the removal of 
the hsematoporphyrin from such urines causes little or no change 
of color, and when this pigment is added to normal urine until on 
spectroscopic examination bands of similar intensity are seen the 
change of tint produced is comparatively slight. In one such case, 
not due to sulphonal, he was able to isolate a purple pigment which 
differed in its properties from any known urinary coloring-matter, 
and to which the color of the urine in question was obviously in the 
main due. Neumeister also states that in sulphonal intoxication an 
iron-containing derivative of haemoglobin occurs in the urine, which 
presents a reddish- violet color and shows a single band of absorption 
in the blue portion of the spectrum immediately bordering on the green. 

Albumin is not present in uncomplicated cases of hsematopor- 
phyrinuria, and the pigment itself does not give the albumin 
reactions. 

To test for hsematoporphyrin, the following procedure may be 
employed : 

Thirty c.c. of urine are treated with an alkaline solution of barium 
chloride. The precipitate, after having been washed with water and 
then with absolute alcohol, is extracted with ordinary alcohol acidu- 
lated with hydrochloric acid, by rubbing in a mortar. The solution 
thus obtained will present a reddish color in the presence of hsema- 
toporphyrin, and its filtrate yields the characteristic spectrum of the 
latter substance — i. e., four bands of absorption, of which two are 
broad and dark and two light and narrow. The former alone are 
characteristic, and frequently the only ones visible. One of these 
extends beyond D into the red portion of the spectrum, while the 
other is situated between b and F. Of the other two bands, one 
may be seen between C and D and the other between J) and E, 
nearer E (Fig. 10). 

Garrod's Method. — To demonstrate the presence of hsematopor- 
phyrin under normal conditions, or when small amounts only are 
present in the urine, Garrod' s method should be employed. To this 
end, several hundred c.c. of urine (500-1500) are treated with a 10 
per cent, solution of sodium hydrate in the proportion of 20 c.c. of 
the alkali solution for 100 c.c. of urine. The precipitated phosphates 
are filtered off and thoroughly washed by repeatedly suspending 
them in water. Should the precipitate be of a reddish color, or if 
it shows the spectrum of hsematoporphyrin in alkaline solution 
when examined on the filter in the moist state, we may con- 



548 THE URINE. 

elude that much hsematoporphyrin is present. In this case it 
is washed until the nitrate is colorless. If traces only are 
present, however, one washing must suffice. The precipitate is 
then treated with alcohol, which is acidified with hydrochloric 
acid to such an extent that the phosphates are entirely dis- 
solved. The resulting solution should not exceed 15 to 20 c.c. in 
volume. This is then examined in a layer, of not less than 3 to 4 
cm. in thickness, for the spectrum of acid hsematoporphyrin, using 
a spectroscope with slight dispersion. The solution is now rendered 
alkaline with ammonia and treated with an amount of acetic acid 
which just suffices to redissolve the precipitated phosphates. On 
shaking with chloroform this extracts the pigment, and the chloro- 
form solution then gives the spectrum of the alkaline hsematopor- 
phyrin, since organic acids do not change the pigment to the form 
which yields the acid spectrum. The residue which remains after 
evaporating the chloroform can finally be washed with water and 
dissolved in alcohol, when a nearly pure solution is obtained, which 
is comparable with a solution of hsematoporphyrin obtained from 
hsematin. 

Precautions : If a preliminary test shows that the urine con- 
tains but little phosphates, a small quantity of calcium phosphate 
in acetic acid is added before the urine is rendered alkaline with the 
sodium hydrate solution. As hsematin and chrysophanic acid are 
also precipitated with the phosphates, their absence must be insured. 
For this reason the urine should contain no rhubarb or senna. 

In conclusion, it may be said that a chromogen of hsematopor- 
phyrin is also usually present in urines containing the free pigments, 
which probably explains why such urines gradually become darker 
on standing. 

Literature. — A complete account of the literature on hsematoporphyrinuria up 
to 1893 is given by E. Zoja, "Su gualche pigmento di alcune urine," etc., Arch, 
ital. di clin. med., 1893, vol. xxxii. p. 63. A. E. Garrod, loc. cit. ; and Cen- 
tralbl. f. inn. Med., 1897, No. 21. Taylor and Sailer, Contributions from the William 
Pepper Laboratory, Philadelphia, 1900, p. 120. O. Neubauer, Arch. f. exper. Path, 
u. Pharmakol., 1900, vol. xliii. p. 455. B. J. Stokvis, " Zur Pathogenese d. Hsemato- 
porphyrinurie," Zeit. f. klin. Med., vol. xxviii. p. 1. East u. Weiss, Berlin, klin. 
Woch., 1896, vol. xxxiii. p. 621. Hammarsten, "Skandin. Arch. f. Physiol.," 1891, 
vol. iii. p. 31. Neumeister, Physiol. Chem., Jena, 1897. Nebelthau, Zeit. f. physiol. 
Chem., 1899, vol. xxvii. p. 324. B. Ogden, Boston Med. and Surg. Jour., 1898. 

Biliary Pigments. — Of the four biliary pigments, viz., bilirubin, 
biliverdin, biliprasin, and bilifuscin, the former alone is met with in 
freshly voided urines, while the others may form upon standing, 
being oxidation-products of bilirubin. The pigment is never found 
in normal urine, and its occurrence may be regarded as a positive 
symptom of disease. 

In health it will be remembered that bilirubin, C^H^N-A, 
formed in the liver from blood-pigment, is eliminated into the small 
intestine, in which it is transformed into hydrobilirubin and largely 



URINARY PIGMENTS AND CHROMOGENS. 549 

excreted as such in the feces, while a small portion is reabsorbed 
into the blood and eliminated in the urine as urochrome or normal 
urobilin. Whenever, then, the outflow of bile into the intestines 
becomes impeded bilirubin is absorbed by the lymphatics and elimi- 
nated in the urine. 

Among the numerous causes which give rise to choluria under 
such conditions may be mentioned obstruction of the biliary ducts, 
and especially of the common duct, referable to simple swelling of 
its mucous membrane, as in the ordinary forms of catarrhal jaun- 
dice. It may also be due to the presence of a biliary calculus, to 
parasites, compression of the duct by tumors of the liver, the gall- 
bladder, the duct itself, and of neighboring structures, and particu- 
larly of the pancreas, stomach, and omentum. Whenever the 
blood-pressure in the liver is lowered, so that the tension in the 
smaller biliary ducts becomes greater than that in the veins, choluria 
likewise results. The icterus occurring under all such conditions 
has been termed hepatogenic icterus, in contradistinction to the form 
observed in cases in which the liver has either totally or partially 
lost the power of forming bile, be this owing to the existence of 
degenerative processes affecting its glandular epithelium, as in cases 
of acute yellow atrophy, or to destruction of red corpuscles 
going on so rapidly and so extensively that the organ is incapable of 
transforming into bilirubin all the blood-pigment which is carried to 
it. This occurs in pernicious anaemia, malarial intoxication, typhoid 
fever, poisoning with arsenious hydride, etc. Icterus neonatorum 
is probably to a certain extent also dependent upon the latter cause. 
To this form the term hematogenic icterus has been applied. In such 
cases the occurrence of bilirubin in the urine can only be explained 
by assuming that a transformation of blood coloring-matter into 
bilirubin has taken place in the blood itself or in other tissues of the 
body. As a matter of fact, it appears to be generally accepted that 
such a transformation can actually occur outside of the liver, as 
the hsematoidin which may be found in old extravasations of blood 
seems to be identical with bilirubin. On the other hand, however, 
the existence of a hematogenic icterus is positively denied, especially 
by Stadelmann. In accordance with his view, it may be demon- 
strated that in cases of pernicious anaemia, malaria, etc., the urine 
does not contain bilirubin, but usually urobilin. In cases of this 
kind which I had occasion to examine, bilirubin was never found. 
Further investigations are necessary to settle this question definitely. 

Usually the presence of biliary pigment may be recognized by 
direct inspection, as urines which contain this in notable amounts 
present a color varying from a bright yellow to a greenish brown. 
Any morphological elements which may occur in the sediment are 
stained a golden yellow, and the same color is imparted to the foam 
of the urine as well as to the filter-paper used in the filtration. At 



550 THE URINE. 

times, however, and particularly in cases in which the icterus is only 
beginning to appear, the presence of bilirubin is not infrequently 
overlooked, and urines containing urobilin in large amounts may be 
similarly mistaken for icteric urines. In doubtful cases, therefore, 
whether icterus exists or not, but in which the urine presents an 
intense yellow color, it is necessary to have recourse to chemical 
tests. A large number of these have been devised for the purpose 
of demonstrating the presence of bilirubin, all of which are fairly 
reliable. Only those will be described which I have examined 
myself and which are especially delicate. 

Smith's Test 1 — Five to 10 c.c. of urine are placed in a test-tube 
and treated Avith 2 or 3 c.c. of tincture of iodine (which has been 
diluted with alcohol in the proportion of 1 : 10) in such a manner 
that the iodine solution forms a layer above the urine. In the pres- 
ence of bilirubin a distinct emerald-green ring is seen at the zone of 
contact. This test can be highly recommended, as it is exceedingly 
simple and not surpassed in delicacy by any other. 

Huppert's Test 2 — Ten to 20 c.c. of urine are precipitated with 
milk of lime (a solution of barium chloride is, perhaps, still more 
convenient), and the precipitate after filtering brought into a beaker 
by perforating the filter and washing its contents into the latter with 
a small amount of alcohol acidulated with sulphuric acid. The 
mixture is boiled, when in the presence of bilirubin the solution 
assumes a bright emerald-green color. Huppert's test is as delicate 
as is that of Smith, but is not so convenient for the needs of the 
practising physician. 

Gmelin's Test (as modified by Rosenbacli)? — The urine is filtered 
through thick Swedish filter-paper, when the latter is removed and 
a drop of concentrated nitrie acid, which has been allowed to stand 
exposed to the air for a short time, is placed upon its inner surface. 
In the presence of bilirubin a prismatic play of colors will be seen 
to occur around the nitric acid spot. 

Gmelin's Test. 4 — The urine is treated with nitric acid, which is 
carried to the bottom of the test-tube by means of a pipette, so as 
to form a layer beneath the urine, when a color-play, as already 
described (page 493), will take place at the line of contact between 
the two fluids ; the green color is the most characteristic. 

In this connection a few words may also be said of the occurrence 
in the urine of biliary acids and cholesterin. 

Biliary Acids. — These may usually be found in the urine whenever 
bile-pigment is present, so that their clinical significance is essen- 
tially the same as that attaching to bilirubin. Their demonstration is, 

1 W. G. Smith, Dublin Med. Jour., 1876, p. 449. 

2 Huppert, Arch. d. Heilk., 1867, vol. viii. pp. 351 and 476. 

3 Eosenbach, Centralbl. f. d. rued. Wiss., 1876, vol. xiv. p. 5. 

4 Tiedemann u. Grnelin, Die Verdauung nach Versuchen, Heidelberg, 1826, 
I. p. 80. 



URINARY PIGMENTS AND CHROMOGENS. 551 

however, attended with such difficulties that the methods devised for 
this purpose may well be omitted at this place (see also page 228). 

Cholesterin. — Cholesterin has never been found in icteric urines, 
and is only rarely seen in other pathological conditions. It has 
been observed in cases of chyluria, fatty degeneration of the kidneys, 
diabetes, in one case of epilepsy, in eclampsia, and in several cases 
of pregnancy. v. Jaksch has noted the presence of cholesterin 
crystals in a urinary sediment in a case of tabes and cystitis. Glin- 
sky records a similar observation. Harley found it repeatedly in 
cases of pyuria. ^Reich states that he found cholesterin crystals of 
the size of a dollar in the urine of a case of chronic cystitis. Hirsch- 
laff found larger quantities in the urine of a case of hydronephrosis ; 
on one occasion 5.8 grammes in 100 c.c. of urine. I have found 
cholesterin crystals in the sediment in a case of acute nephritis. The 
urine was of a dark amber color, cloudy, of an acid reaction, and a 
specific gravity of 1.028. In the sediment numerous hyaline and 
epithelial casts and some red blood-corpuscles were found. Giiter- 
bock described a urinary calculus obtained from the bladder of a 
woman which consisted almost entirely of cholesterin (see also 
Feces). Langgaard noted the presence of the substance in a case 
of chyluria. 1 

Pathological Urobilin. — This pigment should not be confounded 
with the urochrome or normal urobilin described above, to which 
it is closely related, but from which it may be distinguished by 
means of the spectroscope. Gautier states that pathological urobilin 
may be obtained from urochrome by submitting the latter to the 
action of reducing agents ; and, as I have already pointed out, Riva 
and Chiodera obtained a substance from urobilin by the action of 
potassium permanganate, which closely resembles urochrome. It is 
said to be identical with the stercobilin found in the feces, but differs 
from Maly's hydrobilirubin in containing a much smaller percentage 
of nitrogen, viz., 4.11, as compared with 9.22 (Garrod and Hop- 
kins). While its occurrence in the urine is essentially a pathological 
phenomenon, it is at times also met with in normal urine, and 
appears to be derived from a special chromogen, urobilinogen, from 
which it may be set free by the addition of an acid. Both urobilin 
and its chromogen are precipitated by saturating the urine with 
ammonium sulphate, and both are soluble in chloroform. Accord- 
ing to Maly, urobilin is formed by the reduction of bilirubin in the 
intestine, and is then in part resorbed and eliminated in the urine. 
Hayem, on the other hand, proposed the hypothesis that the sub- 
stance originates in a diseased or disordered liver, as bilirubin does 
in the same organ in health, and accordingly he regards the appear- 

1 v. Jaksch. Klinische Diagnostik, 4th etf. p. 339. Glinsky, Maly's Jahresber., 
1894, vol. xxiii. p. 484. Langgard, Virchow's Archiv, vol. lxxxvi. W. Hirschlaff, 
Deutsch. Arch., 1899, vol. lxii. p. 53. 



552 THE URINE. 

ance of much urobilin in the urine as evidence of hepatic insuf- 
ficiency. Others, again, maintain that urobilin is formed in the 
tissues at large either by the reduction of bilirubin or directly from 
the blood-pigment. The first view is notably held by Kunkel, Mya, 
Giarre, and others, while the hematogenous theory is notably rep- 
resented by Gerhardt. Garrod discusses these various hypotheses 
at some length in his most interesting lecture on the urinary pig- 
ments in their pathological aspects, in which he personally inclines 
to the intestinal theory, as now held by Miiller, Schmidt, Esser, and 
others. In a work of this scope it would lead too far to discuss 
the various investigations which lend themselves in support of 
this view, and I can here quote only the following from Garrod' s 
paper : the chief seat of the formation of urobilin (for it is conve- 
nient to employ this term as including both pigment and chromogen) 
is undoubtedly the intestinal canal. This can only be gainsaid by 
denying the identity of the urinary and fecal pigments. The 
quantity normally present in the feces is far larger than that which 
enters the intestine with the bile (when a small amount is found), 
and there is strong evidence that the urobilin in bile is itself of 
intestinal origin. This being so, it is clear that theories other than 
the intestinal and its modifications merely attempt to trace a second 
source for the urobilin of the urine. It is equally clear that the 
substance from which the intestinal urobilin is formed is the bile- 
pigment. Under ordinary conditions the bile-pigment is destroyed 
in its passage along the intestine, and does not appear as such in 
the feces. In its place we find large quantities of urobilin, which 
in its turn disappears when occlusion of the common duct prevents 
the entrance of bile into the intestine. Again, when under certain 
morbid conditions the bile-pigment passes along the intestine unal- 
tered, urobilin is absent from the feces. However, the conversion 
of bilirubin into urobilin is no mere process of reduction, but in- 
volves a much more radical change, with elimination of nitrogen. 
That the change is brought about by bacterial action there is much 
evidence to show. When bile is inoculated with fecal material and 
kept in an incubator a formation of urobilin rapidly takes place, and 
at the same time the bile-pigment diminishes, and ultimately dis- 
appears. 

From its frequent occurrence in febrile urines pathological urobilin 
has also received the name febrile urobilin. It is, however, also 
observed in many other conditions, and especially in cases present- 
ing the so-called hematogenic form of icterus, from which fact, 
indeed, and the usual absence of bilirubin at the same time, this 
form has been termed urobilin icterus. 

Urobilinuria has further been observed in certain hepatic diseases. 
In twelve cases of atrophic and hypertrophic cirrhosis v. Jaksch 
was able to demonstrate the presence of urobilin in every instance, 



URINARY PIGMENTS AND CHROMOGENS. 553 

a point which may at times be of considerable diagnostic importance, 
providing that other causes which are known to lead to urobilinuria 
can be eliminated. I have observed urobilin in a few cases of he- 
patic cirrhosis, chronic malaria, and pernicious anaemia, in all of which 
the skin presented a light icteric hue, and in which bile-pigment 
was absent from the urine. Unfortunately, an examination of 
the blood was not made, and I have hence not been able to con- 
firm the statement of v. Jaksch that bilirubin occurs in the blood 
in almost every case in which urobilin is present in the urine. 
Urobilin has alsa been noted in cases of carcinoma, scurvy, Addi- 
son's disease, haemophilia, in cases of retro-uterine hsematocele, in 
extra-uterine pregnancy, following intracranial hemorrhages, etc. 
According to Bargellini, the degree of constipation in simple atony 
of the bowel is without influence upon the amount of urinary 
urobilin, but he states that in typhoid fever it causes an obvious 
increase ; whereas disinfection or emptying of the large bowel pro- 
duces a notable diminution in the amount. 

Urines rich in urobilin usually present a dark-yellow color which 
is strongly suggestive of the presence of bilirubin ; even the foam 
in such cases may be colored, making the resemblance between the 
two pigments still more complete, v. Jaksch points out, however, 
that urines containing indican in large amounts often likewise 
present a very dark-yellow color, a statement with which my own 
observations are in perfect accord. In every case a more detailed 
chemical examination should hence be made. 

Gerhardt's Test. — If the urine contains much urobilin, which 
the color will indicate, 10—20 c.c. are extracted with chloroform by 
shaking, and the extract treated with a few drops of a dilute solu- 
tion of iodo-potassic iodide. Upon the further addition of a dilute 
solution of sodium hydrate the chloroform extract is colored a yellow 
or yellowish -brown, and exhibits a beautiful green fluorescence ; this 
is even more intense than that noted in the case of normal urobilin. 

Spectroscopic Examination. — This is necessary when Ger- 
hardt's test yields a doubtful result. The urine is then best examined 
as follows : 50 c.c. of urine are extracted in a separation funnel with 
ainyl alcohol, which takes up both the pigment and its chromogen. 
After standing for several hours the urine is allowed to flow away, 
by opening the stopcock, when the alcoholic extract is decanted from 
above, and is treated with a concentrated alcoholic and ammoniacal 
solution of zinc chloride. In the presence of urobilin the liquid 
shows a beautiful fluorescence, and on spectroscopic examination a 
single band of absorption is seen between b and F. In acid solu- 
tions, on the other hand, a single band is likewise obtained between b 
and F, but this extends to the right beyond F, and is much darker. 
Should the urine contain much urobilin, its special extraction is not 
necessary. In such an event the acid urine shows the acid spectrum, 



554 THE URINE. 

while the alkaline band is obtained after the addition of ammonia 
(see also Bang's Test). 

Literatuee. — A. E. Garrod, loc. cit. A. E. Garrod and F. G. Hopkins, " On 
Urobilin," Jour, of Physiol., 1898, vol. xxii. p. 451. Maly, Centralbl. f. d. med. Wiss., 
1871, vol. ix. p. 849. Hayem, Gaz. hebdom., 1887, vol. xxiv. pp. 520 and 534 ; and Gaz. 
des Hop., 1889, vol. lxii. p. 1314. Kunkel, Vircbow's Arcbiv, 1880, vol. lxxix. p. 655. 
Mya, Arch. ital. di clin. med., 1891, vol. xxx. p. 101 ; and Lo Sperimentale, 1896, vol. 1. 
p. 71. Giarre, Ibid., 1895, vol. xlix. p. 89, and 1896, vol. 1. p. 81. F. Miiller,, Schlesische 
Gesellscb. f. vaterland. Kultur, January, 1892. A. Schmidt, Verbandl. d. XIII. Con- 
gress, f. inn. Med., 1895, p. 320. Esser, Untersuchungen uber d. Entstehungsweise d. 
Hydrobilirubins, etc., Diss., Bonn., 1896. Bargellini, Lo Sperimentale, 1892, vol. xlvi. 
p. 119. v. Jaksch, Zeit. f. Heilk., 1895, vol. xvi. p. 48. D. Gerhardt, Zeit. f. klin. Med., 
1897, vol. xxxii. p. 313. 

Melanin and Melanogen. — In cases of melanotic disease it has been 
repeatedly observed that the urine, which usually and probably 
always presents a normal yellow color when voided, gradually 
becomes darker upon exposure to the air, and finally turns black. 
This phenomenon indicates without a doubt that such urines contain 
a chromogen, melanogen, which, upon oxidation, yields the black 
pigment noted in these cases, viz., melanin. As yet, it has not been 
possible to isolate this substance in pure form, and it is, indeed, not 
definitely determined that the black color in such urines is refera- 
ble to a single pigment. Such urines generally contain melanin and 
its chromogen in solution ; deposits of melanin granules by them- 
selves are only occasionally seen, and are not characteristic, as they 
may also be found in cases of chronic malarial intoxication, etc., 
when they may, indeed, be met with in the blood, constituting the 
condition spoken of as melanwmia. 

While the occurrence of melanin in the urine is probably indica- 
tive in most cases of the existence of melanotic tumors, it should 
be stated that this symptom cannot be regarded as pathognomonic, 
as it may be absent in the case of melanotic tumors, and present in 
wasting diseases and inflammatory affections, and may at times, 
though very rarely, even be associated with the presence of non-pig- 
mented growths. Nevertheless, its occurrence should always be 
regarded with suspicion, and, taken in conjunction with other symp- 
toms, will often lead to a correct diagnosis. 

Tests for Melanin and Melanogen. — 1. The presence of 
melanogen may be assumed if upon the addition of ferric chloride 
solution a black precipitate appears in the urine, which is soluble in 
a solution of sodium carbonate and can then be reprecipitated as a 
black or brownish-black powder by means of mineral acids. Instead 
of the ferric chloride barium hydrate may also be used. 

2. A few cubic centimeters of urine are treated with bromine- 
water when in the presence of melanin or melanogen a precipitate is 
obtained, which is yellow at first, but gradually turns black. 

Literature. — T. H. Eiselt, "Die Diagnose d. Pigmentkrebses durch d. Harn," 
Prag. Vierteljahrschr. f. praktische Heilk., 1858, iii. p. 190, and 1862, vol. iv. p. 26. 
Senator, "Leber schwarzen Urin," Charite Annal., 1891. Hoppe-Seyler, Zeit. f. 
physiol. Chem., 1891, vol. xv. p. 179. F. Grohe, " Zur Gesch. d. Melanaemie," Vir- 
chow's Archiv, 1861, vol. xx. p. 306. 



URINARY PIGMENTS AND CHROMOGENS. 555 

Phenol Urines. — The development of a dark-brown or black color 
upon standing is not always due to the presence of melanin, as a 
similar appearance may be noted in cases of poisoning with carbolic 
acid, following the ingestion of salol, hydrochinon, pyrocatechin, 
salicylic acid, etc., in large amounts. The color in such cases is due 
in all probability to the presence of various oxidation-products of 
hydrochinon, and in the last instance possibly to the so-called 
humin-substances. 

The test referred to above will prevent confusion as to the origin 
of the color as far as melanin is concerned, and with the his- 
tory of the case given, moreover, further chemical examination is 
generally unnecessary. In suspected cases of carbolic acid poison- 
ing, however, the mineral as well as the conjugate sulphates should 

be quantitatively determined, when the factor — (see Sulphates) 

will be found greatly diminished. If at the same time other fac- 
tors, which might cause a greatly increased elimination of conjugate 
sulphates, can be excluded, the diagnosis of poisoning with carbolic 
acid or one of its derivatives may be inferred. Salol and salicylic 
acid may be recognized from the fact that such urines when treated 
with a solution of ferric chloride develop a marked violet color which 
does not disappear on standing. The reaction thus differs from that 
obtained with diacetic acid (see also page 573). 

Alkapton. — Urines are at times, though very rarely, seen which, 
like the phenol urines, turn dark on standing, but in which the 
change in color is neither referable to the presence of phenol or its 
derivatives, nor to melanin. Such urines are of a normal color when 
passed, but gradually turn reddish brown upon exposure to the 
air. Treated with a small amount of alkali, this change occurs 
almost immediately. Fehling's solution is reduced on the applica- 
tion of heat, while bismuth is not affected. Ammoniacal silver 
solution is reduced in the cold, and a temporary bluish-green color 
develops when the urine is treated with a ferric salt. The fermenta- 
tion test is negative, and examination with the polarimeter shows 
that the substance in question is not glucose. With phenylhydrazin 
no osazon is formed. 

Bodeker, who first observed a urine of this kind, termed the sub- 
stance giving rise to the reactions just described alkapton, and sub- 
sequently expressed the belief that his alkapton might possibly have 
been pyrocatechin. Subsequent investigators succeeded in isolating 
substances from such urines which have been variously termed pyro- 
catechuic acid, urrhodinic acid, glucosuric acid, uroleucinic acid, and 
uroxanthinic acid. Baumann and Wolkow later were able to iso- 
late homogentisinic acid in pure form from the urine of such cases, 
and expressed the belief that some of the substances obtained by 
previous observers were in reality the same. Since that time this 



556 THE URINE. 

acid has also been found by Garrod, Ogden, Stange, Stier, and others. 
There is reason to believe, however, that the reaction is not always 
due to one and the same substance. 

Of the origin of alkapton little is known. Baumann expressed 
the opinion that homogentisinic acid might be derived from tyrosin, 
and that the condition is referable to the activity of special micro- 
organisms in the upper portions of the intestines. As a matter of 
fact the amount of homogentisinic acid can be very materially in- 
creased by the administration of tyrosin, and Mittelbach has shown 
that if the substance is given in frequently repeated and small doses 
almost the entire amount reappears in the urine as homogentisinic 
acid. Tyrosin, however belongs to the para-series, while homogen- 
tisinic acid is an or£/io-compound, so that the transformation of tyro- 
sin into homogentisinic acid cannot be a direct process, and it has 
accordingly been questioned whether Bauman's view regarding the 
origin of alkapton is correct. There is evidence indeed to show that 
homogentisinic acid does not originate in the intestines, viz., is not a 
product of bacterial activity. It has thus been found that the alkap- 
tonuria does not cease during starvation, aud that a restriction of the 
putrefactive processes in the intestines by means of oil of turpentine, 
a kefir diet, and the administration of /9-naphthol does not lead 
to a diminished elimination of homogentisinic acid. It has never 
been found in the feces, moreover ; and Garrod has shown that after 
inoculation of common bouillon, meat-juice, or ty rosin broth with 
alkaptonuric feces homogentisinic acid is not formed. Embden 
observed that when an alkaptonuric individual took homogentisinic 
acid by the mouth a far larger portion appeared in the urine than 
when the same substance was administered to a healthy individual, 
which suggests that the alkaptonuria may be referable to impair- 
ment of the normal processes of oxidation. 

The prevailing view at the present time is accordingly that 
alkaptonuria is a metabolic anomaly comparable to glucosuria and 
cystinuria ; but, unlike glucosuria, it can scarcely be regarded as an 
expression of a pathological process. It may, of course, occur in 
individuals, suffering from disease, and has thus been observed in 
connection with glucosuria, in acute gastro-intestinal catarrh, in 
phthisis, acute miliary tuberculosis, in one case of brain tumor, 
carcinoma of the prostate, etc. More frequently the condition is 
accidentally discovered in apparently healthy individuals, and has 
repeatedly been confounded with glucosuria owing to the positive 
reduction test with Fehling's solution. 

Garrod, from an analysis of all the reported cases, concludes that 
the condition is nearly always congenital. In 32 known instances 
which were presumably congenital, 19 occurred in seven families. 
One family contained 4 alkaptonurics, three others 3, and the re- 
maining three 2 each. In fully 60 per cent, of the cases, it appears 



URINARY PIGMENTS AND CHROMOGENS. 557 

from Garrod's studies, the parents of alkaptonurics were first cousins. 
There is thus far only one known instance in which the anomaly has 
been transmitted by an alkaptonuric father to his son. 

The condition commonly persists through years and perhaps a life- 
time. It may also occur as a transitory abnormality, however, as is 
apparent from the case of Hirsch, in which the condition persisted 
for three days, and the case of Geyger, in which the alkaptonuria was 
observed on only two days. A few observers further report on the 
occurrence of alkaptonuria shortly preceding death. 

The amount of homogentisinic acid eliminated in the twenty-four 
hours is variable, but usually large. Baumann found an average 
elimination of 4.6 grammes ; the largest amount eliminated in 
twenty-four hours was 6 grammes. In Meyer's case, a child one 
and one-half years old, 3.3 grammes were passed pro die. Larger 
quantities are obtained after a liberal diet of meats than with a vege- 
table diet. By the administration of tyrosin the amount can be 
artificially increased ; in one of Baumann's cases an elimination of 
14 grammes in the twenty-four hours could be thus produced. 
Phenylpropionic acid and benzoic acid do not cause an increased 
excretion of homogentisinic acid. After the administration of phenyl- 
acetic acid, on the other hand, the power of reduction of the urine is at 
times increased, and following the ingestion of 10 grammes of phenyl- 
amidoacetic acid Embden noticed an increase in the power of rotation 
of 36.5 per cent. This, of course, suggests that the acid in question 
may be concerned in the production of increased amounts of homo- 
gentisinic acid, but actual transformation has not as yet been observed. 
A notable increase is also produced by phenylalanin. 

To isolate homogentisinic acid from alkapton urines, and to deter- 
mine its amount, Baumann's method may be employed. The col- 
lected urine of twenty-four hours is acidified with 250 c.c. of a 
12 per cent, solution of sulphuric acid and extracted three times 
with an equal volume of ether. The ethereal extract is evaporated 
to a syrup. The crystals which separate out on standing are dis- 
solved in 250 c.c. of water. This solution is brought near the boil- 
ing-point, and is then treated with 30 c.c. of a neutral lead acetate 
solution (1 : 5) and rapidly filtered. In the filtrate the lead salt 
crystallizes out in transparent needles and prisms. This is then 
decomposed with hydrogen sulphide and the filtrate carefully evap- 
orated on a w^ater-bath until the fluid begins to darken, when it is 
further concentrated in the vacuum to the point of crystallization. 
The resulting prismatic crystals are almost colorless and transparent. 
They melt at a temperature of 146.5°— 147° C, and are readily 
soluble in water, alcohol, and ether, and are almost insoluble in 
chloroform, benzol, and toluol. A solution of the acid, which may 
thus be isolated in pure form, presents the same characteristics as 
the urine from which it was obtained. 



558 THE URINE. 

The following method, suggested by Garrod, may also be employed, 
and has the advantage of greater simplicity. 

Gaerod's Method. — The urine itself is heated nearly to boiling 
without any preliminary treatment, and for each 100 c.c. of urine at 
least 5 or 6 grammes of solid neutral lead acetate are added. 

As soon as the acetate is dissolved, the bulky gray precipitate 
which forms is removed by filtration, and the filtrate, which has a 
pale-yellow color, is allowed to stand for twenty-four hours in a cool 
place. If the urine be very rich in homogentisinic acid, or if the 
flask containing it be placed upon ice, minute acicular crystals, which 
are almost colorless, quickly form ; but as a rule crystallization 
does not commence until several hours have elapsed. The crystals 
are then much larger, are grouped in stars or rosettes, and are more 
deeply colored. 

In summer weather it would probably be desirable to start the 
crystallization by artificial cooling ; but although the process is greatly 
accelerated at a low temperature, the total yield is not materially 
increased. 

If formation of the crystals be long delayed, the liquid may be 
warmed again and more lead acetate added. 

After the lapse of twenty-four hours crystals cease to form, even 
when the liquid is placed upon ice. 

The crystalline product so obtained is lead homogentisinate. When 
the crystals are dissolved in hot water the solution assumes a deep- 
brown color with alkalies ; it reduces Fehling's solution readily with 
the aid of heat, and yields a transitory deep-blue color with a 
dilute solution of ferric chloride. From the lead salt free homo- 
gentisinic acid may be obtained by decomposing it with hydrogen 
sulphide. 

Estimation. — Entirely satisfactory methods for the quantitative 
estimation of homogentisinic acid in the urine are not available. 
The following method, however, is sufficiently accurate for clinical 
purposes : 50 c.c. of urine are treated with 15 grammes of ammo- 
nium chloride, which should be brought into solution by shaking, in 
a stoppered graduate. After standing for about twelve hours to 
allow the uric acid to separate out the solution is filtered and an 
accurately measured portion of the filtrate titrated with a decinor- 
mal ammoniacal solution of silver nitrate. The titration is con- 
tinued until a further reduction of the silver solution does not occur, 
which is ascertained by acidifying a few drops of the filtered mixt- 
ure with hydrochloric acid, when in the presence of free silver a 
turbidity referable to silver chloride occurs. Accuracy within nar- 
rower limits than J c.c. is scarcely possible, as the turbidity refer- 
able to silver chloride can only be recognized within 0.2-0.3 c.c. 
According to Baumann, 240-245 c.c. of the silver solution repre- 
sent 1 gramme of homogentisinic acid. 



PLATE XX. 



■ "' r '~ ■ 



Ehrlieh's Diazo-Reaetion, as modified, by the author. 
The orange color in the lower portion of the test tube may 
be obtained in any urine; the dark, carmine ring indicates 
the presence of the reaction in a well-pronounced degree; 
the colorless zone above is intended to indicate the am- 
monia that has been added. 



URINARY PIGMENTS AND CHROMOGENS. 559 

Literature. — Bodeker, Annal. d. Chemie u. Pharmakol., 1861, vol. cxvii. p. 98. 
Baumann u. Wolkow, Zeit. f. physiol. Chem., 1891, vol. xv. p. 228. Stier, Berlin, klin. 
Woch., 1898, vol. xxxv. p. 185. Embden, Zeit. f. physiol. Chem., 1893, vol. xvii. p. 182, 
and vol. xviii. p. 304. Ogden, Zeit. f. physiol. Chem., 1895, vol. xx. p. 280. Futcher, 
N, Y. Med. Jour., 1898, vol. lxvii. p. 69. Garrod, Jour. Physiol., 1899, vol. xxiii. p. 
512 ; and Med.-Chir. Trans. Boval Soc, vol. lxxxii. p, 367. E. Meyer, Deutsch. Arch., 
vol. lxx. Hefte 5 u. 6. F. Wittelbach, Ibid., 1901, vol. lxxi. p. 50. 

Blue Urines. — Blue urines are sometimes seen, the color of which 
is due to indigo formed from urinary indican, in all probability 
within the urinary passages. Their occurrence can only be regarded 
as a medical curiosity. One case of this kind is reported by 
McPhedran and Goldie, 1 in which after direct extraction of the urine 
with ether only a faint reaction was obtained on further examina- 
tion, and which probably was referable to incomplete previous 
extraction. Formerly, when indigo was employed in the treatment 
of epilepsy, blue urines were frequently seen. At the present time, 
when methylene-blue is occasionally used in the treatment of malaria 
and chyluria, this pigment is found in the urine. 

Green Urines. — Green urines have also been described ; the cause 
of the color, however, has not been definitely ascertained. 

Pigments referable to Drugs. — Certain drugs may also cause 
changes in the normal color of urine, and in doubtful cases inquiry 
in this direction should be made. It has been pointed out that car- 
bolic acid, hydrochinon, pyrocatechin, and salol cause the appearance 
of a dark-brown color, and that after the administration of indigo 
and methylene-blue blue urines are voided. Santonin, rheum, and 
senna color urines a bright yellow, so that they may resemble icteric 
urines in appearance. The yellow color in such cases is changed to 
an intense red by the addition of an alkali, and, if ammoniacal fer- 
mentation is going on at the same time in the bladder, the patient 
may believe himself to be suffering from hematuria. The red color 
thus produced is due to the action of the alkali upon chryso- 
phanic acid. When urines containing copaiba are treated with 
hydrochloric acid a red color results, which changes to violet upon 
the application of heat. During the administration of potassium 
iodide, or the use of iodine in any form, a dark mahogany color is 
obtained when the urine is treated with nitric acid. In doubtful 
cases Stokvis' modification of Jaffe's test for indican should be em- 
ployed, when in the presence of an iodide the chloroform assumes a 
beautiful rose-red color. 

For the detection of other drugs and poisons in the urine the 
reader is referred to special works. 

Ehrlich's Reaction. — Under certain pathological conditions, and 
especially in typhoid fever, a chromogen may be present in the urine, 
which, when treated with diazo-benzene-sulphonic acid and am- 
monia, imparts a distinct red color to the urine, varying from eosin 
to a deep garnet-red (Plate XX.). This reaction, which is generally 

1 Trans, Assoc Am. Phys., 1901. 



560 THE URINE. 

spoken of as Erhlich's reaction, or the diazo-readion, was at one 
time regarded as pathognomonic of typhoid fever. Subsequent 
examinations, however, have shown that it may also be present in 
other diseases. Michaelis, who has made an exhaustive study of 
this question, divides into four groups the diseases in which the reac- 
tion has been observed. In the first group, comprising diseases of 
the nervous system, chronic diseases of the heart and kidneys, 
malignant tumors, etc., the reaction is rarely seen. When present, 
it usually indicates a secondary infection. The second group in- 
cludes those diseases in which the reaction is almost always present, 
namely, typhoid fever and measles. In the diseases of the third 
group it is often, though not invariably, observed. Under this 
heading are classed scarlet fever, erysipelas, pneumonia, diphtheria, 
pyaemia, acute miliary tuberculosis, etc. The fourth group comprises 
pulmonary tuberculosis, and includes acute caseous pneumonia. 

The value of Ehrlich's reaction in typhoid fever was at first overesti- 
mated, but is at present certainly underestimated. I have personally 
studied this problem with great care, and after many years' experience 
maintain, as I did years ago, that the test is a most important 
diagnostic aid in the disease in question. As a general rule the 
reaction is present as early as the fifth or sixth day, and may persist 
into the third week ; it then disappears, but may reappear when a 
relapse occurs. Excepting in children, its absence from the fifth to 
the ninth day usually indicates a mild case. This rule, however, is 
not without exception, and I have seen a case of typhoid fever in 
which notwithstanding exceedingly high temperatures (106.5° at 6 
A. m.) the reaction was not obtained until the beginning of the 
third week, and then persisted for only a few days. When the 
reaction is continuously present after the third week I am inclined 
to suspect acute tuberculosis. It may be present as early as the 
fourth day of the disease. 

In paratyphoid as in typhoid fever the reaction is also fairly con- 
stant. 

Of late much attention has been paid to the occurrence of 
Ehrlich's reaction in pulmonary phthisis. As a result of his inves- 
tigations Michaelis concludes that its presence in such cases indi- 
cates either that the process is very extensive or that it will 
progress very rapidly, and that the prognosis is grave. A cure, 
he believes, is impossible, and improvement, if any, only tempo- 
rary. Clemens notes that of 100 cases of phthisis which ended 
fatally 87 showed the diazo reaction ; Riitimeyer obtained positive 
results in 85 cases out of 106 which died. Of 13 cases of acute 
tubercular pneumonia Frankel and Troje found a positive reaction 
in 11. Grundriss states that in his fatal cases the reaction was 
present without exception. Similar results have been obtained by 
Cnopf, See, Goldschmidt, and others, Michaelis himself reports 



URINARY PIGMENTS AND CHROMOGENS. 561 

that of 111 cases of phthisis which were admitted to the Berlin 
Charite with well-marked reaction 80 died in the hospital, 13 were 
discharged unimproved, 3 were transferred to other hospitals, and 
15 left improved. In other words, of these 111 cases a fatal result 
was known to have occurred in 72 per cent. Stadelmann states 
that of 38 other cases with positive reaction 28 died in the hospital 
— i. e., about 75 per cent. The subsequent fate of the remaining 
cases was not ascertained ; but we may well assume that of these 
at least 50 per cent, died, so that we may formulate the general rule 
that a fatal result may be anticipated in about 85 per cent, of all 
cases of phthisis in which a positive reaction is obtained. Michaelis, 
moreover, suggests that the end may be expected to occur within 
six months from the time at which a persistent Ehrlich's reaction 
is established. Exceptions occur, but the above is the rule. In 
Koch's institute at Berlin patients presenting the diazo reaction 
are not treated with tuberculin, as such cases are generally regarded 
as hopeless (Brieger). 1 

In tubercular peritonitis the diazo reaction is found in about 25 
per cent, of all cases. 

As regards the frequency of occurrence of the reaction in diph- 
theria, it appears from the observations of Bivier 2 and others 
that it is decidedly uncommon. Of his own 118 cases, and 44 
additional onces collected from the literature, only 10 gave a posi- 
tive result ; and of these, 4 should be eliminated as they occurred 
in complicated cases, so that the reaction was absent in about 97 
per cent. 

In the scarlatiniform erythema due to serum treatment the reac- 
tion is apparently absent, while in true scarlatina it is fairly 
common. Including a number of cases collected from the lit- 
erature Bivier found a positive reaction in 41 cases out of 73. 
He concludes that in the differential diagnosis between the two 
conditions scarlatina may be affirmed, if the reaction is positive, 
while if negative there is strong presumptive evidence against the 
disease. 

In measles a positive reaction was obtained in 75 of 85 cases. 

The reaction is possibly due to the presence of alloxyproteinic 
acid. 3 

As the preparation of chemically pure, crystalline diazo-com- 
pouncls is a difficult process, Ehrlich uses sulphanilic acid, which, 
when treated with nitrous acid in a nascent state, gives rise to the 
formation of diazo-benzene-sulqhonic acid, as is shown by the 
equations : 

1 Discussion on Tuberculosis, Michaelis, Deutsch. med. Woch., 1901. v. b. p. 211. 

2 Eivier, These de Paris, 1898. 

3 Bondzynski u. Panek, Berlin, d. deutsch. chem. Ges., 1903, vol. xxxv. p. 2951. 

36 



562 THE VRINE. 

(1) NaN0 2 + HC1 = NaCl -f KN0 2 . 

(2) C 6 H,< +HN0 2 = C 6 H 4 < ^N + 2H 2 0. 

Para-amido- Diazo-benzene- 

benzene-sulphonic acid. sulphonic acid. 

This is the active principle in the mixture employed. 

Other compounds may, of course, also be used, such as meta-amido- 
benzene-sulphonic acid, ortho- and para-toluidin-sulphonic acid, etc. ; 
but of all these, Ehrlich found the common sulphanilic acid the most 
convenient. Two solutions, which must be kept in separate bottles, 
are employed. The one is a 5 per cent, solution of hydrochloric 
acid, to which sulphanilic acid is added in the proportion of 1 
gramme for each 100 c.c. The other is a 0.5 per cent, solution of 
sodium nitrite. 

The two solutions are mixed in the proportion of 40 to 1 im- 
mediately before using. A few cubic centimeters of urine are then 
treated with an equal volume of the reagent, the mixture is shaken 
and rendered alkaline with ammonium hydrate. This is best 
allowed to flow down the sides of the tube, so as to form a layer 
above the mixture. At the junction of the two fluids a colored ring 
will now be observed. With urines which do not contain the 
chromogen this will be a more or less distinct orange, while in its 
presence a red color is obtained. The intensity of this color may 
vary from eosin to a deep garnet-red. If the mixture is now agi- 
tated and the reaction is positive, the foam will likewise be colored 
red, and upon pouring the solution into a porcelain basin containing 
much water a beautiful salmon color is obtained, even if only traces 
of the chromogen are present. Carried out in this manner no 
question will arise as to the presence or absence of the reaction. 
Ehrlich states that on standing a green sediment forms in the 
alkalinized mixture, and he regards this sediment as especially char- 
acteristic. My experience has been that this becomes manifest only 
when the color-reaction is well pronounced, and I am inclined to attach 
more importance to the salmon color obtained upon copious dilution. 
With normal urines this is never obtained, and it can still be seen 
when inspection of the fluid in the test-tube would leave in doubt. 

The older method of Ehrlich I have abandoned, as the test 
just described is simpler, and, in my experience, just as reliable. 
He advised the addition of about 50 c.c. of absolute alcohol to 10 
c.c. of urine, subsequent filtration, and examination of the filtrate, 
as just described. 

Greene states that if 1 part of the sodium nitrite solution is 
added to 100 instead of 40 parts of the sulphanilic acid solution, a 
positive reaction is no longer obtained in cases of croupous pneu- 
monia and of pulmonary tuberculosis, while in typhoid fever the 



URINARY PIGMENTS AND CHROMOGENS. 



563 



Cases Tested. 



Typhoid fever 

Malarial fever 

Tetanus 

Acute miliary tuberculosis . . 
Joint tuberculosis ..... 
Pulmonary tuberculosis . . . 

Septicaemia 

Ulcerative endocarditis . . . 
Secondary syphilis . . . . . 

Erysipelas 

Scarlatina 

Measles * . . . 

Carcinoma 

Pneumonia 

Rheumatism, chronic .... 

Rheumatism, acute 

Diphtheria 

Diarrhoea 

Appendicitis 

Albuminuria of pregnancy . 

Chronic nephritis 

Cystitis 

Urethritis, specific 

Oxaluria and lithsemia . . . 

Pleurisy 

Pyaemic abscess of lung . . . 
Tuberculosis of prostate . . . 
Necrosis of long bones . . . 

Rotheln 

Syphilis (third stage) .... 

Alcoholic neuritis 

Hysteria 

Epilepsy 

Leg ulcer, varicose 

Fractures, long bones .... 

Fracture, skull . 

Burns, severe 

Gunshot wounds, aseptic . . 
Morphin poisoning .... 

Sciatica 

Cirrhosis, hepatic „ . . . . 

Simple enteritis 

Angioneurotic oedema . . . . 

Endometritis 

Pericarditis 

Meningitis ........ 

Vulvitis and vaginitis, specific 
Orchitis, gonorrhceal .... 

Valvular heart-disease . . . 
Quinsy and tonsillitis .... 

Normal urines 

Varicella 

Typhoid relapse ...... 

Gastric ulcer 

Acute bronchitis 

Chronic constipation .... 



Total 
Number. 



64 
4 
2 
3 
4 

16 
4 
1 
4 
2 
3 
2 
4 

11 

10 
5 
3 
4 
3 
6 

19 
2 
7 

11 
5 
1 
3 
2 
1 
5 
3 
6 
2 
7 
5 
2 
2 
2 
1 
3 
2 
3 
2 
3 
1 
1 
2 
1 
7 
3 

30 
1 
3 
2 
3 
7 



Reaction. 



Present. Absent 



315 



61 1 




2 
3 





2 
1 






































3 






3 
4 

2 
3 
4 

14 
1 
1 
4 
2 
3 
2 
2 

10 

10 
5 
3 
4 
3 
6 

19 
2 
7 

11 
5 
1 
3 
2 
1 
5 
3 
6 
2 

7 
I 

5 

2 
2 
2 
1 
3 
2 
3 
2 
3 
1 
1 
2 
1 
7 
3 
30 
1 

2 
3 
7 



95 per cent. 



564 THE URINE. 

reaction occurs with the same intensity. It is thus possible that the 
test may be still further modified, and become even more valuable. 
On page 563 are given some of the results which Greene obtained 
with this method. 

While in the absence of the chromogen, as I have already stated, 
a more or less pronounced orange color is usually obtained, excep- 
tions have been noted. Ehrlich thus records that in urines contain- 
ing biliary coloring-matter an intensely dark, cloudy discoloration 
occurs at times, which upon boiling is changed to a well-marked 
reddish violet. In rare instances of ulcerative endocarditis, hepatic 
abscess, and intermittent fever, Ehrlich further observed an intense 
yolk-yellow color, which was even imparted to the foam. 

Of interest is the observation of Burghart, that after the adminis- 
tration of tannic acid, gallic acid, and certain iodine preparations, 
Ehrlich' s reaction disappears from the urine. But, as Burghart 
himself suggests, it is likely that this inhibitory effect is not exerted 
upon the diazo-forming substance, but upon the reagents employed. 
Other factors, which may prevent the occurrence of Ehrlich's reac- 
tion, in pulmonary tuberculosis at least, are the occurrence of renal 
complications (albuminuria). Naphthalin, after its administration 
by the mouth, according to my experience causes a reaction, the 
color of which corresponds exactly to that of the diazo reaction. 

Literature.— Ehrlich, Zeit. f. klin. Med., 1882, vol. v. p. 285; Charite Anna]., 
1883, vol. viii. p. 28, and 1886, vol. xi. p. 139. Goldschmidt, Munch, med. Woch., 
1886, vol. xxxiii. p. 35. Biitimeyer, Corresp. Blatt. f. Schweizer Aerzte, 1890, vol. xxvi. 
Greene, Med. Eecord, Nov. 14, 1896. C. E. Simon, Johns Hopkins' Hosp. Bull., 1890. 
J. Friedenwald, N. Y. Med. Jour., 1893. M. Michaelis, Berlin, klin. Woch., 1900, p. 
274; and Deutsch. med. Woch., 1899, p. 156. J. E. Arneill, Am. Jour. Med. Sci., 
1900, p. 296. 

Ehrlich's Dimethylamidobenzaldehyde Reaction. — Ehrlich has 
shown that under various pathological conditions a fine cherry-red 
color develops on shaking a specimen of urine with a- few drops of 
dimethylamidobenzaldehyde in acid solution, and that the resulting 
pigment can be in part extracted with chloroform, and almost 
entirely so with epi- or dichlorhydrin. With normal urines a simi- 
lar reaction can be obtained, but it is much less intense, and if done 
at ordinary temperatures a distinct red color does not develop. On 
heating, however, it appears, and can likewise be extracted with 
epichlorhydrin. Of the nature of the substance which gives rise to 
the red color nothing definite is known. 

The occurrence of the reaction in disease has been studied by 
Clemens, Kozickowsky, and myself. I can summarize my own 
results as follows : 1. A direct reaction, of pathological grade, does 
not occur in health. 2. A positive reaction is most commonly 
obtained in cases of tuberculosis. 3. It may also be seen in non- 
tubercular cases, both febrile and non-febrile. 4. It is not depend- 
ent upon the presence of the body which gives rise to the diazo 



URINARY PIGMENTS AND CHROMOGENS. 565 

reaction. 5. For its production elevation of temperature, gastro- 
intestinal disturbances, and cyanosis are not essential. 6. Common 
to all cases seems to be an increased katabolism of the tissue albu- 
mins. 

My positive results include cases of pulmonary tuberculosis, 
tuberculosis of the hip-joint, pneumonia, typhoid fever, appendi- 
citis, embarras gastrique, icterus, malignant endocarditis, empyema, 
oesophageal carcinoma, and a remarkable instance of traumatic neu- 
rosis, in which a loss of weight of from sixty to seventy-five pounds 
had occurred. 

My list of negative cases, on the other hand, includes, first of 
all, a large number of normal or supposedly normal individuals ; in 
addition, cases of normal labor, neurasthenia, hysteria, diabetes, 
aortic aneurism, myelogenous leukaemia, lymphatic leukaemia, acute 
nephritis (scarlatinal), simple diarrhoea, morphinism, valvular dis- 
ease, phthisis (stationary), diphtheria (before and after the use of 
antitoxin), typhoid fever, cases of abortion, appendicitis, influenza, 
chronic nephritis, cystitis, pyelitis (calculous), measles, tuberculosis 
of the hip-joint, cystic kidney, carcinoma of the kidney, tonsillitis, 
acute and chronic bronchitis, pneumonia, icterus, tuberculous perito- 
nitis, general erythema, varicocele, following various operations, 
such as nephrorrhaphy, removal of pus tubes, operations for vesico- 
vaginal fistula, fistula in ano, and suspension of uterus. Examina- 
tion of a urine containing cystin and diamins was also negative. A 
comparison of the negative with the positive cases will show at 
once that not all cases of pulmonary tuberculosis, tuberculous hip- 
joint disease, pneumonia, typhoid fever, appendicitis, and icterus 
give a positive result. So far as tuberculosis is concerned, however, 
it appears that the reaction is more likely to occur in the actively 
progressive cases than in those which are more or less stationary. 
It was also noted that the positive cases almost all gave a positive 
diazo reaction, while in the negative cases this was not obtained. 
Exceptions, however, may also occur. 

In my personal examinations I employed a 2 per cent, solution 
of dimethylparamidobenzaldehyde in equal parts of water and con- 
centrated hydrochloric acid. A few cubic centimeters of urine in a 
test-tube are treated with from 5 to 10 drops of the reagent; the 
mixture is set aside or agitated for a few minutes and the color then 
noted. Normal urines usually turn a greenish yellow, or the normal 
color merely becomes intensified. At times a dark amber color 
develops, though this is less common in health, unless the urine is 
brought to the boil before the reagent is added. In this way it is a 
common experience to meet with moderate or dark amber tints. 
With these reactions, however, I have not occupied myself, and, 
like Clemens and Koziczkowsky, I have only noted the reac- 
tion as positive when a distinct cherry-red color developed, 



566 THE URINE. 

either immediately on adding the reagent or after agitation or 
standing. 

Literature. — Ehrlich, Med. Wocb., 1901, No. 15, Clemens, Deutsch. Arch., 1901, 
vol. lxxi. p. 168. Koziczkowsky, Berl. med. Woch., 1902, vol. xxxix. No. 44. Simon, 
Am. Jour, Med. Sci., 1903, vol. cxxvi. p. 471. 

CONJUGATE SULPHATES. 

In addition to indoxyl (see Indican), skatoxyl, phenol, paracresol, 
and pyrocatechin occur in the urine in combination with sulphuric 
acid. 

Skatoxyl. — Skatoxyl results from the skatol formed during the 
process of intestinal putrefaction, as indoxyl is derived from indol, 
and is partly eliminated in the urine as skatoxyl sulphate. Clini- 
cally it is of little interest, as the amount excreted is very small, and 
it is not necessary to enter into a further consideration of its chemi- 
cal properties or mode of detection at this place (see Feces). 

Phenol. — Phenol, according to Brieger, occurs only in very small 
amounts in human urine, the usual phenol reactions being largely 
referable to paracresol. Normally, about 0.03 gramme is eliminated 
in the twenty-four hours, but in pathological conditions much larger 
quantities may be found. Remembering the origin of phenol, it is 
clear that an increased elimination may be observed whenever putre- 
factive processes are going on in the tissues and cavities of the body, 
or whenever there is an increase in the degree of intestinal putre- 
faction, though in the latter condition the indican is usually the only 
conjugate sulphate that is found increased. In peritonitis, diph- 
theria, erysipelas, scarlatina, empyema, pulmonary gangrene, putrid 
bronchitis, etc., an increased elimination of phenol is commonly 
seen. Important from a diagnostic standpoint, further, is the fact 
that in uncomplicated cases of typhoid fever no increase is observed, 
while this is common in tubercular meningitis. 1 The largest amounts, 
of course, are seen in cases of poisoning with carbolic acid or one of 
its derivatives. 

As the quantitative estimation of phenol is too complicated for the 
purposes of the general practitiouer, Salkowski's qualitative test is 
here also described. From the intensity of the reaction certain con- 
clusions may be drawn as to the amount present. It is especially 
serviceable in cases of suspected poisoning with carbolic acid. 

Salkowski's Test. — About 10 c.c. of urine are boiled in a test- 
tube with a few cubic centimeters of nitric acid, and, on cooling, 
treated with bromine-water. The development of a pronounced 
turbidity or the occurrence of a precipitate indicates the presence 
of an increased amount of phenol. 

1 A Strasser, " Ueber d. Phenolausscheidung bei Krankbeiten," Zeit. f, klin. Med., 
vol. xxiv. p. 543. Brieger, Zeit. f. klin. Med., 1881, vol. iii. p. 468. Kast u. Boas, 
Munch, med. Wocb., 1888, vol. xxxv. p. 55. 



CONJUGATE SULPHATES. 567 

Quantitative Estimation. — Principle. — When potassiuui-phenyl 
sulphate is treated with hydrochloric acid, phenyl sulphate results, 
which further takes up one molecule of water, giving rise to the 
formation of sulphuric acid and phenol, according to the following 
equations : 

/).C 6 H 5 /O.C 6 H 5 

(1) S0 2 < +HC1= KC1 +S0 2 < 

X)K X)H 

/O.C 6 H 5 /OH 

(2) S0 2 < + H 2 = S0 2 < +C 6 H 5 .OH. 

From the action of bromine-water upon phenol a yellowish-white 
crystalline precipitate of tribromophenol results : 

C 6 H 5 .OH + 6Br = 3HBr + C 6 H 2 Br 3 .OH. 

As 331 (molecular weight) parts by weight of tribromophenol 
correspond to 94 (molecular weight) parts by weight of phenol, the 
amount of the latter contained in a certain volume of urine is readily 
determined according to the equation 

331 : 94 : : x : y ; and y == — = 0.28398 x, 
y, if 331 

in which x indicates the weight of the tribromophenol found in the 
amount of urine employed, and y the corresponding quantity of 
phenol. 

Method. — From 500 to 1000 c.c. of urine are treated with one-fifth 
of an equivalent amount of dilute hydrochloric acid (1 : 4), and dis- 
tilled so long as a specimen of the distillate is rendered cloudy upon the 
addition of bromine- water (1 : 30), the specimens used fo this purpose 
being carefully preserved. The total quantity of the filtered dis- 
tillate, together with the specimens which have been set aside, is now 
treated with bromine-water, shaking the mixture after each addition 
of the reagent until a permanent yellow color results. Beyond this 
point further addition is beset with danger, as compounds will be 
formed w r hich contain more bromine, the presence of which would 
indicate a smaller amount of phenol than that actually contained in 
the urine. After two or three days the precipitate is collected on a 
filter which has been dried over sulphuric acid, washed with water 
containing a trace of bromine, and then dried over sulphuric acid 
and weighed. 

Pyrocatechin. — Urines containing pyrocatechin, like those con- 
taining hydrochinon (see above), darken upon standing, though 
presenting a normal color when voided. 



568 THE URINE. 



ACETONE. 



The amount of acetone which may be found in the urine under 
normal conditions varies between 0.008 and 0.027 gramme, and is 
greatly influenced by the character of the diet. Whenever the car- 
bohydrates are withdrawn the quantity rapidly increases, and reaches 
its maximum about the seventh or eighth day. At this time from 
200 to 700 mgrms. may be eliminated in the twenty-four hours. 
If, then, carbohydrates are again added to the diet, the acetonuria 
soon disappears. This result is not reached, however, if fats are sub- 
stituted for the carbohydrates. The acetonuria is greatest when but 
little albuminous food and no carbohydrates at all are ingested, and 
during starvation the same amounts are essentially found. There 
can hence be no doubt that acetone is derived from proteid material. 
Increased amounts are accordingly found whenever, as in fevers, the 
various cachexias, in conditions associated with inanition, etc., large 
quantities of circulating albumin are broken down, or whenever car- 
bohydrates are not furnished in sufficient amount. 1 

Some other observers have recently attempted to show that the 
fat is the source of the acetone, but it seems to me that even though 
fat may be its source it is certainly not its only source, and there 
is indeed more evidence available to show that acetone may be 
derived from carbohydrates, if we are to consider sources outside 
of the albumins. 2 Blumenthal and Neuberg have succeeded in 
obtaining acetone directly from gelatin by oxidation, so that its 
possible origin from this source at any rate can be regarded as 
established. 

Most important is the diabetic form of acetonuria, and it may be 
stated, as a general rule, that the diagnosis of diabetes mellitus is 
justifiable whenever sugar and notable quantities of acetone are 
found in the urine. The amount of acetone, moreover, stands in a 
direct relation to the intensity of the disease, the maximum excretion 
being usually observed toward the fatal end. 3 Whether or not this 
form of acetonuria can always be explained upon the basis given 
above remains an open question. There can be no doubt, however, 
that the threatening symptoms which are so commonly associated 
with a greatly increased elimination of acetone will often disappear 
when carbohydrates are administered in large amounts. It is certain, 
moreover, that diabetic coma is more apt to occur when the old- 
fashioned plan of excluding carbohydrates entirely from the dietary 
of diabetic patients is adopted. Hirschfeld 4 suggests that in every 

1 v. Jaksch, Ueber Acetonurie u. Diaceturie, Hirschwald, Berlin, 1885. Eosenfeld, 
Centralbl. f. inn. Med., 1895, vol. xv. Waldvogel, " Zur Lehre von der Acetonurie," 
Zeit. f. klin. Med., vol. xxxviii. p. 506. 

2 C. Neuberg u. F. Blumenthal, Zeit. f. chem. Phys. u. Pathol., 1902, vol. ii. p. 238. 

3 v. Jaksch, Zeit. f. klin. Med., 1885, vol. x. p. 362. Lorenz, Ibid., 1891, vol. xix. p. 19. 

4 F. Hirschfeld, " Beobacbtungen uber d. Acetonurie u. das Coma diabeticum," 
Zeit. f. klin. Med., vol. xxviii. p. 176, and vol. xxxi. p. 212. 



ACETONE. 569 

case of diabetes the excretion of acetone be carefully followed, and 
that large amounts of carbohydrates be administered whenever the 
acetonuria approaches a dangerous extent. This agrees with my 
experience. 

Of the febrile diseases in which acetonuria has been observed 
may be mentioned typhoid fever, pneumonia, scarlatina, measles, 
acute miliary tuberculosis, acute articular rheumatism, and septi- 
caemia. In those of short duration, on the other hand, even if the 
fever is high, as in acute tonsillitis, intermittent fever, the hectic 
fever of phthisis, etc., an increased elimination of acetone is rarely 
observed. In the continued fevers the acetonuria is largely referable 
to the character of the diet, as carbohydrates are usually excluded 
entirely, and I have repeatedly observed that a return to the normal 
occurred as soon as sugar was administered in amounts varying from 
50 to 100 grammes. 

In certain nervous and mental diseases, as in general paresis, mel- 
ancholia, following epileptic seizures, and in tabes, acetonuria is fre- 
quently observed. During the second stage of general paresis in- 
creased amounts are indeed quite constantly found, but Hirschfeld 
is probably correct in stating that the psychotic form of acetonuria 
is largely referable to improper feeding. 

In the primary diseases of the stomach, and notably in carcinoma, 
acetonuria is frequently observed, and it is possible that the acetone 
in these cases is ..to some extent at least formed in that organ directly 
from the proteids ingested. The fact that in carcinoma acetone may 
be observed at a time when marked loss of flesh has not as yet 
occurred, and that larger amounts of acetone may be found in the 
stomach than in the urine, is certainly in favor of this view. 1 

An enterogenic form of acetonuria has further been described, and 
it has been urged that in these cases the acetone is referable to the 
formation of unusually large amounts of fatty acids. Acetonuria 
of this type is also observed following the ingestion of fatty acids 
as such (alimentary form). The cases of so-called asthma acetoni- 
cum probably belong to this class. 2 

Acetonuria has further been observed early in the course of acute 
phosphorus poisoning, and may persist throughout, apparently 
without being an index of the severity of the case. 

After chloroform narcosis the condition is also not uncommon. 

Tests for Acetone. — Legal's Test. 3 — This test may be applied 
to the freshly voided urine, but is not conclusive. Several cubic 
centimeters of urine are treated with a few drops of a strong solution 
of sodium nitroprusside and sodium hydrate ; the mixture assumes 
a red color, which rapidly disappears, and in the presence of acetone 

1 H. Lorenz, loc. cit. 

2 Waldvogel u. Hagenberg, "Ueber alimentare Acetouurie," Zeit. f. klin. Med., 
1900, vol. xiii. p. 443. 

3 Le Xobel, Arch. f. exper. Path. u. Pharmakol., 1884, vol. xviii. p. 9. 



570 THE URINE. 

is replaced by a purple or violet red when acetic acid is added. As 
a rule, it is better to distil the urine (500-1000 c.c.) after the addition 
of a little phosphoric acid (1 gramme pro liter), and to employ the 
first 10-30 c.c. of the distillate for one or more of the following tests. 

Lieben's Test. 1 — A few cubic centimeters of the distillate are 
treated with several drops of a dilute solution of iodo-potassic 
iodide and sodium hydrate, when in the presence even of traces of 
acetone a precipitation of iodoform in crystalline form occurs, which 
may be readily recognized by its odor when the solution is heated. 

Reynolds' Test. 2 — A few cubic centimeters of the distillate are 
treated with a small amount of freshly precipitated yellow mercuric 
oxide. This is prepared by precipitating a solution of mercuric 
chloride with an alcoholic solution of sodium hydrate. If acetone 
is present, a black color, due to the formation of mercuric sulphide, 
will result in the clear filtrate upon the addition of a few drops of 
ammonium sulphide. 

Stock's Test (modified by Frohner). — A crystal of hydroxylamin 
hydrochlorate is dissolved in about 5 c.c. of the distillate ; the 
solution is treated with hypochlorite of calcium solutiou and ex- 
tracted with a little ether. A blue color is obtained, which can 
still be discerned in the presence of but 0.001 gramme of acetone. 
The reaction is proof positive of the presence of a ketone, but is 
for this reason also obtained with diacetic acid. 

As originally proposed by Stock, 10 c.c. of the .liquid to be ex- 
amined are treated with 1 drop of a 10 per cent, solution of hydrox- 
ylamin, 1 drop of a 5 per cent, solution of sodium hydrate, and a 
larger drop of pyridin, after which the ether (about 1 c.c.) is added, 
as also bromine-water while shaking, until the ether has assumed a 
yellow tint. On the subsequent addition of hydrogen peroxide the 
yellow changes to blue. 

Denniges' Test (as modified by Oppenheimer). 3 — The reagent is 
prepared as follows : 20 grammes of concentrated sulphuric acid 
are poured into 100 c.c. of distilled water, when 5 grammes of 
freshly prepared yellow mercuric oxide (see Reynolds' test) are 
added. The mixture is allowed to stand for twenty-four hours and 
is then ready for use. 

This reagent is added to about 3 c.c. of urine, drop by drop, 
until the precipitate which is thus formed no longer disappears on 
stirring. When this point is reached a few more drops are added. 
After two to three minutes the precipitate is filtered off. The clear 
filtrate is further treated with about 2 c.c. of the reagent, and 3-4 
c.c. of a 30 per cent, solution of sulphuric acid, and boiled for a 
minute or two, or, still better, placed in a vessel with boiling water. 

1 Taniguti u. Salkowski, Zeit. f. physiol. Chem., 1890, vol. xiv. p. 476. 

2 Gunning, Jour, de Pharmacol, et de Chim., 1881, vol. iv. p. 30. 

3 Oppenheimer, Berlin, klin. Woch., 1899, p. 828. 



ACETONE. 571 

Id the presence of an abundant amount of acetone a copious white 
precipitate forms immediately ; while in the presence of traces only 
(less than 1 : 50000), a slight cloud develops on standing for several 
minutes. The precipitate is almost entirely soluble in an excess of 
hydrochloric acid. 

If albumin is present, the urine becomes turbid at once when the 
reagent is added. In that case the test is continued as described, 
attention being directed to the coarser precipitate which occurs later. 
To such urines large amounts of the reagent must be added, the idea 
being to precipitate everything that can be precipitated with the 
reagent, before heating. 

It will be observed that Denniges' test is much simpler than the 
tests already described, and Oppenheimer claims that it is as delicate 
as that of Lieben, viz., giving a well-pronounced reaction with a 
dilution of 1 : 20000, and being still discernible with a dilution of 
1 : 60000. As diacetic acid yields acetone when treated with 
mineral acids, a positive result is always obtained when this is pres- 
ent. But as diacetic acid is usually found only in association with 
acetone, this fact does not lessen the value of the test, and is an 
error, moreover, which is common to the other tests as well. 

Quantitative Estimation of Acetone. — For the purpose of 
estimating the amount of acetone the method of Messinger, as 
modified by Huppert, is now employed, and is greatly to be preferred 
to the older procedure of v. Jaksch. 1 

Principle. — The method is based upon the observation of Lieben 
that acetone gives rise to the formation of iodoform when treated 
with iodine in an alkaline solution. If, then, a solution of acetone is 
treated with a known amount of iodine, it is a simple matter to 
determine the quantity present by retitrating the iodine which was 
not used in the formation of iodoform. 

Solutions required : 

1. Acetic acid (50 per cent, solution). 

2. Sulphuric acid (12 per cent, solution). 

3. Sodium hydrate solution (50 per cent.). 

4. A decinormal solution of iodine. 

5. A decinormal solution of sodium thiosulphate. 

6. Starch solution (see page 252). 
Preparation of the solutions : 

1. The decinormal solution of iodine is prepared as described 
elsewhere (see page 251). 

2. As the molecular weight of sodium thiosulphate — Na 2 S 2 3 -f 
5H 2 — is 248, a decinormal solution of the salt would contain 24.8 
grammes to the liter. This quantity is dissolved in about 950 c.c. 
of distilled water, and brought to the proper strength by titration 

1 See Neubaueru. Vogel, Analyse des Haras, 9th ed., p. 470. 



572 THE URINE. 

with a decinormal solution of iodine. As 1 c.c. of the thiosulphate 
solution should correspond to 1 c.c. of the iodine solution, the neces- 
sary amount of water which must be added to the former is then 
determined. 

Method. — One hundred c.c. of urine, or less if much acetone is 
present, as determined by Legal' s test, are treated with 2 c.c. of the 
acetic acid solution and distilled until seven-eighths of the total 
amount have passed over. The distillate is received in a retort 
which is connected with a bulb-tube containing water. As soon 
as seven-eighths of the urine have distilled over, a small amount 
of the distillate of the remainder is tested for acetone according 
to Lieben's method. Should a positive reaction be obtained, it 
will be necessary either to repeat the entire process with less urine, 
diluted to about 200 c.c, or to add about 100 c.c. of water to the 
residue and to distil until all the acetone has passed over. The 
distillate is then treated with 1 c.c. of the sulphuric acid and redis- 
tilled. The addition of the acetic acid and of the sulphuric acid, 
respectively, serves the purpose of holding back phenol and am- 
monia. Should the first distillate contain nitrous acid, moreover, 
which is recognized on the addition of a little starch paste contain- 
ing a trace of potassium iodide, when the solution turns blue, the 
acid is removed by adding a little urea. The second distillate is re- 
ceived in a bottle provided with a well-ground glass stopper, and 
holding about 1 liter. The distillate is then treated with a carefully 
measured quantity of the one-tenth normal solution of iodine, — 
about 10 c.c. for 100 c.c. of urine, — and sodium hydrate solution 
until the iodoform separates out. To this end, a slight excess of the 
solution must be added. Should ammonia be present, a blackish 
cloud will be observed at the zone of contact of the sodium hydrate 
and the iodine solution, and it will be necessary to repeat the entire 
process. The bottle is closed and shaken for about one minute. 
The solution is then acidified with concentrated hydrochloric acid, 
when the mixture assumes a brown color if iodine is present in 
excess. If this does not occur, more of the iodine solution must 
be added, and the process repeated until an excess is present. The 
excess is then retitrated with the thiosulphate solution until the fluid 
presents a faint-yellow color. A few cubic centimeters of starch 
solution are now added, and the titration continued until the last 
trace of blue has disappeared. The number of cubic centimeters 
employed in the titration is finally deducted from the total amount 
of the iodine solution added, and the result multiplied by 0.976. 
The figure thus obtained indicates the amount of acetone contained 
in the 100 c.c. of urine, in mgrms., as 1 c.c. of the thiosulphate 
solution is equivalent to 1 c.c. of the iodine solution, or to 0.967 
mgrm. of acetone. 



DI ACETIC ACID. 573 

DIACETIC ACID. 

The occurrence of diacetic acid in urine must always be regarded 
as abnormal. Its pathological significance is identical with that 
of acetonuria. It is met with especially in diabetes, in various 
digestive diseases, and in febrile di.-eases. In the continued fevers 
of childhood it is almost constantly present. 

Gerhardt's Test. — In order to demonstrate the presence of 
diacetic acid a few cubic centimeters of urine are treated with 
a strong solution of ferric chloride added drop by drop. A pre- 
cipitate of phosphates is filtered off, when more of the iron solu- 
tion is added to the filtrate. If now a Bordeaux-red color appears, 
another portion of the urine is boiled and similarly treated. If 
in the second test no reaction is obtained, a third portion of the 
urine is treated with sulphuric acid and extracted with ether. A 
positive reaction, when the ethereal extract is tested with ferric 
chloride, the color disappearing upon standing for twenty-four to 
forty-eight hours, will indicate the presence of diacetic acid, par- 
ticularly if the urine is rich in acetone. With Gerhardt's test a 
negative reaction is obtained even though diacetic acid be present, 
if the patient has been taking salol, viz., when salicylic acid is 
beino; excreted bv the urine. In such case the urine should be 
filtered through animal charcoal, which retains the salicylic acid, but 
does not interfere with the diacetic acid. 

Arnold's Test. — Two solutions are employed, viz., a solution 
of para-amido-aceto-phenon and a 1 per cent, solution of sodium 
nitrite. The first is prepared by dissolving 1 gramme of para- 
amido-aceto-phenon in from 80 to 100 c.c. of distilled water, and 
adding hydrochloric acid drop by drop until the solution, which at 
first is yellow, becomes colorless ; an excess, however, should be 
avoided. Immediately before using, the two solutions are mixed in 
the proportion of two to one. A few cubic centimeters of the reagent 
are then treated with an equal volume of urine, and a few drops of 
ammonia added. Thus treated, all urines give a more or less marked 
brownish-red color on shaking ; and if much diacetic acid is present, 
an amorphous reddish-brown sediment is thrown down. A small 
amount of the colored solution is then placed in a conical glass and 
treated with an excess of concentrated hydrochloric acid (10—12 c.c. 
for each 1 c.c). In the presence of diacetic acid the mixture assumes 
a beautiful purplish- violet color. 

According to Arnold, the test is more delicate than that of Ger- 
hardt, and does not respond with acetone or oxybutyric acid. With 
bilirubin and the common antipyretics, as well as salicylic acid, no 
reaction is obtained. Highly colored urines should first be filtered 
through animal charcoal. 

According to Lipliawski, the following modification of Arnold's 



574 THE URINE. 

test is even more sensitive : two solutions are employed, viz., a 1 per 
cent, solution of para-amido-aceto-phenon and a 1 per cent, solution 
of potassium nitrite. Six c.c. of the first solution and 3 c.c. of the 
second are added to an equal volume of urine, together with a drop 
of concentrated ammonia. The mixture is shaken until it assumes 
a brick-red color. From 10 drops to 2 c.c, according to the 
amount of diacetic acid present, are treated with 15—20 c.c. of 
concentrated hydrochloric acid (sp. gr. 1.19), 3 c.c. of chloroform, 
and 2-4 drops of an aqueous solution of ferric chloride. The tube 
is closed with a cork and gently agitated (so as to avoid emulsifica- 
tion), when after one-half to one minute a beautiful and very charac- 
teristic violet tinge results if diacetic acid is present. In its absence 
the color is yellowish or slightly reddish. The violet color per- 
sists for a long time. Salicylic acid, phenacetin, antipyrin, phenol, 
and other drugs are without disturbing influence upon the reaction. 
Allard states that both Arnold's test and that of Lipliawski 
give a positive result also with acetone, when this is present to the 
extent of more than 1 per cent. 

Literature. — v. Jakscli, Ueber Acetonurie u. Diaceturie, loc. cit. Idem., Zeit. f. 
Heilk., 1882, vol. iii. p. 34. Sclirack, Jahrbuch f. Kinderbeilk, 1889, vol. xxix. p. 411. 
V. Arnold, Wien. kliu. Woch., 1899, p. 541. 



OXYBUTYRIC ACID. 

The fact that in some cases of diabetes an excessive elimination 
of ammonia was observed led to the belief that there must be 
present an unknown acid ; this was shown to be /3-oxybutyric acid. 
The occurrence of this acid in the urine of diabetic patients is of 
great clinical interest, not only from the standpoint of diagnosis, but 
also of prognosis and treatment. Its presence may always be re- 
garded as indicating a severe type of the disease, and when asso- 
ciated with marked acetonuria and diaceturia as indicating the 
possible occurrence of coma. 

According to Herter, the condition of diabetic coma is preceded 
by a period of days, weeks, or months, in which there is a large 
excretion of /3-oxybutyric acid (20 grammes or more in twenty-four 
hours), and in which the nitrogen of ammonia is largely increased. 
The same writer states that patients whose urines show or have 
shown a large excretion of organic acids are in danger of devel- 
oping diabetic coma ; but the nitrogen of ammonia may temporarily 
rise as high as 16 per cent., and yet coma may be delayed for more 
than seven months. The persistent excretion of more than 25 
grammes of /9-oxybutyric acid indicates impending coma. Impor- 
tant also is the observation that while as a general rule the appear- 
ance of large amounts of organic acids is associated with the presence 



OXYBUTYRIC ACID. 575 

of much sugar, a constant relation between the two does not exist. 
There may thus be much sugar and little or no acid in the urine, or 
there may be much acid and little sugar. 

The presence of oxybutyric acid may be inferred in diabetic 
urines if after fermentation a rotation of the plane of polarization 
to the left is observed. 

Quantitative Estimation (according to Darmstaedter). — The method 
is based on the decomposition of the /3-oxybutyric acid with the for- 
mation of a-crotonic acid and the estimation of the latter. This 
decomposition takes place according to the equation : 

CH 3 .CHOH.CH 2 .COOH = CH 3 .CH.CH.COOH + H 2 0. 

0-oxybutyric acid. Crotonic acid. 

100 c.c. of urine are rendered feebly alkaline with sodium carbo- 
nate and evaporated on a water-bath almost to dryness. With 
the aid of 150-200 c.c. of sulphuric acid (50-55 per cent.) the 
residue is transferred to a litre flask, which is closed with a 
doubly perforated stopper. Through the one aperture a drip-tube 
passes, while a bent glass tube passes through the other to a con- 
denser. Heat is applied, at first mildly, so as to avoid foaming ; 
then vigorously. Water is allowed to enter through the drip- 
tube as fast as the distillate passes over. The distillation is inter- 
rupted when from 300 to 350 c.c. have been obtained, which usually 
takes from two to two and one-half hours. The distillate is ex- 
tracted two or three times with ether. The ether is distilled off, 
the residue heated for a few minutes on a sand-bath to 160° C. in 
order to drive off any fatty acids that may be present, and then dis- 
solved on cooling with 50 c.c. of water. The solution is filtered, 
and the filter washed with a little water. The aqueous solution of 
the crotonic acid is now titrated wdth a decinormal sodium hydrate 
solution, using phenolphthalein as an indicator. 1 c.c. of the soda 
solution corresponds to 0.0086 gramme of crotonic acid. The cor- 
responding amount of oxybutyric acid is obtained by multiplying 
by 1.21. Sugar does not interfere with the process. 

If it is only desired to prove the presence of oxybutyric acid in 
the urine, this method can also be conveniently employed. The cro- 
tonic acid is obtained from the ethereal extract, and recognized by 
its melting-point, 72° C. If necessary, it can be purified by solu- 
tion in water and re-extraction with a small amount of ether and 
subsequent evaporation, viz., distillation of the ether. 

Literature. — v. Jaksch, Ueber Acetonurie u. Diaceturie, loc. cit. H. Wolpe, 
Arch. f. exper. Path. u. Pharmakol., 1886, vol. xxi. p. 131. Herter, "The Acid In- 
toxication of Diabetes in its Eelation to Prognosis," Jour, of Exper. Med., 1901, vol. 
v. p. 617, E. Darmstaedter, Zeit. f. phys. Cheni., 1903, vol. xxxvii. p. 355. 



576 THE URINE. 

CROTONIC ACID. 

As has just been shown, crotonic acid is a derivative of oxybu- 
tyric acid. Its presence in the urine as such has not as yet been 
established, and it is likely that statements to the contrary are based 
upon findings of the acid in the distillate, especially when the dis- 
tillation has been carried on after the addition of sulphuric acid to 
the urine. But even in the absence of a free acid a small amount 
of crotonic acid results from oxybutyric acid on boiling. 

LACTIC ACID. 

Sarcolactic acid is normally absent from the urine, but is met with 
in pathological conditions, and particularly in hepatic diseases, as 
the liver is normally concerned in the decomposition of lactic acid and 
of the lactates that have been ingested with the food. As has 
been pointed out, moreover, there is evidence to show that by far 
the greatest portion of the nitrogen eliminated from the body reaches 
the liver as ammonium lactate, and is here synthetically transformed 
into urea. As a consequence, lactic acid appears in the urine when- 
ever, as in phosphorus poisoning, acute yellow atrophy, etc., an 
extensive destruction of the hepatic parenchyma occurs, and the 
formation of urea is consequently impaired. In such cases the 
elimination of lactic acid is associated with an increased excretion 
of ammonia. The same will occur when, owing to insufficient oxy- 
genation of the blood, the power of oxidation on the part of the 
liver is interfered with. We accordingly find lactic acid in the urine 
in the chronic ansemias, in cases of poisoning with carbon monoxide, 
in association with the various forms of circulatory and respiratory 
dyspnoea, in cases of epilepsy immediately after the attack, following 
excessive muscular exercise, as in soldiers after forced marches, etc. 

In order to test for lactic acid, the urine is evaporated on a water- 
bath to a thick syrup and extracted with 95 per cent, alcohol. This 
is decanted off after twenty-four hours, evaporated to a syrup, acidi- 
fied with dilute sulphuric acid, and extracted with ether so long as 
this presents an acid reaction. The ether is then distilled off and 
the residue dissolved in water. This solution is treated with a few 
drops of a solution of basic lead acetate, filtered, the excess of lead 
removed by means of hydrogen sulphide, and the filtrate evaporated 
to dryness on a water-bath, when the lactic acid will remain behind 
as a slightly yellowish syrup. This is then dissolved in a little 
water, the solution is saturated with zinc carbonate, and boiled. 
Zinc lactate will separate out upon evaporation, especially if a little 
alcohol is added, and may be recognized by the form of its crystals, 
viz., small prisms. These crystals are lsevorotatory, soluble in alco- 
hol (1 : 1100), and contain two molecules of water of crystallization, 



VOLATILE FATTY ACIDS. 577 

which is lost at 105° C, so that the loss of weight after heating to 
this temperature must correspond to 12.9 per cent. 

Literature. — O. Minkowski, " Ueber den Einfluss d. Leberextirpation auf d. 
Stoffwecbsel," Arch. f. exper. Path. u. Pharmakol., vol. xxi. p. 41; and "Ueber 
Ursache d. Milchsaureausscbeidung nach Leberextirpation," Ibid., vol. xxxi. p. 214. 
G. Colosanti u. R. Moscatelli, " Ueber d. Milcbsiiuregebalt d. rnenschlichen Harns, 
Ibid., vol. xxvii. p. 158. Jnouyeand Saiki, " Lactic Acid after Epileptic Attacks," Zeit. 
f. physiol. Chem., 1903, vol. xxxvii. p. 203. 

OXYAMYGDALIC ACID. 

Schultzen and Riess * discovered an acid in the urine of patients 
who had died from acute yellow atrophy to which they gave the 
formula C 8 H 8 4 . They regard it as oxyamygdalic acid and suppose 
it to be derived from ty rosin, which was also found, according to 
the equation : 

C 9 H n N0 3 + 30 = C0 2 + NH 3 + C 3 H 8 4 . 

Very curiously it was not found in cases of phosphorus-poisoning, 
but only in acute yellow atrophy. As in this disease there is 
coincidently with the rapid parenchymatous destruction much ex- 
travasation of blood, Nencki has suggested that the acid in question 
may possibly be derived from blood-pigment, especially as Kiister 
obtained from hsematoporphyrin an acid which has the formula 
C 8 H 8 5 , and which thus only differs from the product of Schultzen 
and Riess by a plus of one atom of oxygen. 

VOLATILE FATTY ACIDS. 

The term lipaciduria is applied to the elimination of volatile 
fatty acids in the urine. This occurs under normal conditions, but 
may be much more marked in disease. With an ordinary diet the 
degree of lipaciduria corresponds to from 50 to 80 c.c. y-g- normal 
sulphuric acid. In febrile conditions, according to v. Jaksch and 
Rokitansky, there is an increase, which runs parallel to the height 
of the temperature. Rosenfeld, however, has shown that this is, 
strictly speaking, not correct, and that an increase is only observed 
in those febrile states in which resorption of breaking down albu- 
minous material is taking place, as in cases of tonsillar abscess, 
septic diphtheria, putrid bronchitis, and empyema, and in general in 
association with all suppurative processes and hemorrhages within 
the body. Especially high values are found during convalescence 
from pneumonia, during the first days following crisis. This is no 
doubt owing to a resorption of the exudate, and is associated with 
an increased elimination of nitrogen. Immediately before the crisis 
it is common to meet with very low values — 20 c.c. — as compared 

1 O. Scbultzen u. L. Eiess, Annalen d. Charite Krankenhauses zu Berlin, 1869, 
vol. xv. 

37 



578 THE URINE. 

with 100-240 c.c. during convalescence. These observations, as 
Rosenfeld has pointed out, may be of marked value in the diagnosis 
of obscure accumulations of pus. 

A marked decrease in the amount of fatty acids is noted in 
uncomplicated cases of erysipelas and scarlatina (30-50 c.c), in 
measles, diphtheria, and, as I have already indicated, in pneumonia 
preceding active resorption of the exudate (20-40 c.c). 

According to some observers, the amount of fatty acids in the 
urine may be regarded as an index of the degree of carbohydrate 
fermentation in the intestinal tract. Under normal conditions this 
may be the case, but in disease the question is probably more 
complicated. 

The acids in question are formic acid, acetic acid, butyric acid, 
and propionic acid. They may be isolated as described in the 
chapter on the Feces. 

For their quantitative estimation it will suffice to distil a given 
volume of urine with sulphuric acid and to titrate the distillate 
with y 1 ^- normal sodium hydrate solution. The results are expressed 
in terms of the number of c.c. of ^tt normal sulphuric acid corre- 
sponding. 250 c.c of the urine, which must be fresh or preserved 
with chloroform, are distilled with 50 c.c. of dilute sulphuric acid 
until 200 c.c. have passed over. The residue is diluted with 200 
c.c. of water and the distillation continued as before. In this 
manner the danger that some hydrochloric acid may pass over is 
avoided, but it is well to make sure of this by testing with silver 
nitrate. 

The method is exact ; traces of benzoic acid are included, but in 
man these can be neglected. 

Litekatuee. — v. Jaksch, Zeit. f. klin. Med., 1886, vol. xi. p. 307 ; and Zeit. f. 
physiol. Chem., 1886, vol. x. p. 536. 

FAT. 

Under strictly normal conditions the urine contains no fat, while 
variable amounts may be found in disease. When present in large 
quantities, so that it is possible to recognize it with the naked eye, 
the condition is termed lipuria. Such cases, however, are rare, and 
the diagnosis should only be made when it is possible to exclude an 
accidental contamination of the urine. Smaller quantities of fat, 
recognizable only with the microscope, are much more common, and 
are indeed quite constantly observed whenever fatty degeneration of 
the renal epithelial cells, of pus-corpuscles, or of tumor-particles is 
taking place in the urinary tract. The fat-droplets may then be found 
floating in the urine or attached to or imbedded in any morphological 
elements that may be present. Lipuria may also occur when ab- 
normally large quantities of fat are circulating in the blood. It is 



FERMENTS. 579 

thus observed after the administration of cod-liver oil in large quan- 
tities, following oil inunctions, in cases of fracture of the long bones 
with extensive destruction of the bone-marrow, in cases of eclampsia, 
as also in such diseases as diabetes mellitus, chronic alcoholism, 
phthisis, obesity, leukaemia, in certain mental diseases, in affections 
of the pancreas and heart, etc. 

The term chyluria or galacturia has been applied to a condition 
in which a turbid urine presenting the macroscopical appearance of 
milk is excreted. Upon microscopical examination it may be de- 
monstrated that the turbidity in such cases is owing to the presence 
of innumerable highly refractive globules of fat, which may be 
removed by shaking with ether. Of morphological constituents, 
leucocytes are occasionally encountered in large numbers. Red 
blood-corpuscles are also seen at times, and when present in large 
numbers impart a rose color to the urine. Fibrinous coagula are 
often observed when the urine has stood for some time, and the 
entire bulk of urine may even become transformed into a gelatinous 
mass. Albumin is present in most cases in the absence of other 
constituents pointing to renal disease, such as tube-casts and renal 
epithelial cells. Leucin, tyrosin, and cholesterin may also at times 
be found, particularly the latter. It has been quite generally 
accepted that chyluria is due to the presence of the Filaria sanguinis 
hominis ; but while filarise are undoubtedly present in the blood in 
the majority of instances, and may also be present in the urine, it 
has been demonstrated that cases occur in which filariasis does not 
exist, and Gotze expressed the opinion that chyluria may be owing 
to a distinct anatomical lesion affecting the renal parenchyma. 

Literature. — Lipuria : Sehiitz, Prag. ined. Woch., 1882, vol. vii. p. 322. Ebstein, 
Arch. f. klin. Med., 1879, p. 115. Chyluria: Huber, Virchow's Archiv, 1886, vol. 
cvi. p. 126. Kossbach-Gotze, Verhandl. d. Congr. f. inn. Med., 1887, vol. vi. p. 212. 
Brieger, Zeit. f. physiol. Chem., 1880, vol. iv. p. 407. Grim, Langenbeck's Archiv, 
1885, vol. xxxii. p. 511. 

FERMENTS. 

Ferments may be demonstrated in every urine, both under physio- 
logical and pathological conditions. Pepsin (viz., a proteolytic fer- 
ment) is said to be absent in cases of typhoid fever, carcinoma of 
the stomach, and possibly also in nephritis. In order to demonstrate 
its presence, a small flake of boiled fibrin is placed in the urine, and 
after several hours removed to a 2 to 3 pro mille solution of hydro- 
chloric acid. The pepsin, if present, will be deposited upon the 
fibrin and effect digestion of the latter in the hydrochloric acid 
solution. Diastase, a milk-curdling ferment, a fat-splitting ferment, 
and a ferment causing decomposition of urea into carbon dioxide 
and ammonia, have also been observed. 

It is noteworthy that the fat-splitting ferment was encountered 



580 THE URINE. 

in a case of hemorrhagic pancreatitis, and it has been suggested that 
its presence may possibly be of value in the diagnosis of the disease. 
Opie, who reports the case, demonstrated its presence by the method 
of Castle and Loevenhart. Only a small amount of urine was 
obtained. This was neutralized with potassium hydroxide and 
divided into two portions. To one portion were added a few drops 
of carefully purified ethyl butyrate together with a small quantity of 
litmus solution. The second portion used as a control was boiled in 
order to destroy the ferment if present, and ethyl butyrate added. 
Both specimens were kept at 37° C. ; at the end of twenty-four 
hours the unboiled specimen had acquired a well-marked acid reac- 
tion, while the control specimen was little if at all changed. 

Since the diagnosis of acute lesions of the pancreas is difficult and 
at times impossible the demonstration of the constant occurrence of 
the ferment under such circumstances would be of great importance. 
Future investigations in this direction are urgently needed. 

Literature. — Opie, Johns Hopkins Hospital Bull., 1902, vol. xiii. p. 117 ; Castle 
and Loevenhart, Am. Chem. Jour., vol. xxiv. 

GASES. 

Every urine contains a small amount of gases, notably carbon 
dioxide, oxygen, and nitrogen, which may be withdrawn by means 
of an air-pump. 

Under pathological conditions hydrogen sulphide is at times also 
found, constituting the condition known as hydroikionuria. In 
some instances this is referable to a diffusion of the gas into the 
bladder from neighboring organs or accumulations of pus ; but this 
is rare. In others an abscess has ruptured into the bladder, or a direct 
communication exists between it and the bowel. Under such con- 
ditions it can, of course, not be surprising that hydrogen sulphide 
together with other products of albuminous putrefaction are elimi- 
nated in the urine. More commonly, however, the hydrothionuria 
occurs idiopathically, and is then referable to the action of certain 
micro-organisms. This can be readily demonstrated by adding a 
few cubic centimeters of such urine to normal urine, when upon 
standing the formation of hydrogen sulphide may be demonstrated 
in the normal specimen. The common organisms, however, which 
cause ammoniacal decomposition apparently have no part in this 
process, and the formation of the hydrogen sulphide may be ob- 
served before ammoniacal decomposition has set in and while the 
reaction is yet acid. If a small amount of ordinary decomposing 
urine, moreover, is added to fresh normal urine, no hydrogen sul- 
phide is as a rule produced. The character of the organisms in 
question is variable ; sometimes micrococci are found, at other times 
bacilli, and in still other instances both. Besides being capable of 



PTOMAINS. 581 

producing hydrogen sulphide from the sulphur bodies of the urine, 
some of them also cause the formation of ammonium carbonate in 
dilute solutions of urea. 

The source of the hydrogen sulphide in cases of hydrothionuria 
is in most cases probably the so-called neutral sulphur, but it is pos- 
sible that the oxidized sulphur is at times also attacked. Very in- 
teresting is the fact that in cystinuria, in which the neutral sulphur 
is more or less increased, hydrothionuria is commonly observed. 
Its occurrence in such cases is indeed so frequent that I am in- 
clined to suspect cystinuria, although crystals of cystin are not 
found in the sediment. Further work in this direction, however, 
is needed, and especially to determine the relative frequency with 
which the two conditions are associated. 

In a few recorded instances the hydrothionuria accompanied 
indigosuria, viz., the presence of free indigo-blue in the urine ; and 
this Miiller has likewise shown to be referable to the action of cer- 
tain micro-organisms (see page 627). One case of this kind I saw 
several years ago, but made no examination for the presence of 
cystin. 

Owing to the well-known poisonous effect of hydrogen sulphide 
upon the blood, it is well in every case to ascertain whether its 
formation occurs in the bladder, or whether it takes place only, on 
standing. The formation of hydrogen sulphide in decomposing urines 
containing albumin is, of course, common, and should not be con- 
fused with the idiopathic hydrothionuria here described. 

The chemical test for hydrogen sulphide is very simple : a strip 
of filter-paper is moistened with a few drops of sodium hydrate 
and lead acetate solution and clamped into the neck of the bottle 
containing the urine. After a variable length of time, in some 
instances immediately, in others only after twelve to twenty-four 
hours, a discoloration of the paper will be observed, varying from 
a grayish brown to black according to the amount present. When 
this is large it is, of course, also recognized by its characteristic odor. 

Literature. — F. Miiller, " Schwefelwasserstoff ini Harn," Berlin, klin. Woch., 
1887, Xos. 23 and 24. Kosenheim u. Gutzmann, Deutsch. med. Woch., 1888, No. 10. 
Kahler, Prag. med. Woch., 1888, No. 50. 

PTOMAINS. 

Numerous researches have shown that traces of toxic alkaloidal 
substances may be encountered in the urine under the most diverse 
pathological conditions, and may be present even in health. Of 
the nature of these bodies, however, little is known. Thudichum 
claims to have isolated three distinct basic substances from normal 
urine, which he has termed reducin, pararedudn, and arom'in. 
Pouchet and Mme. Eliacbeff, working in Gautier's laboratory, have 
likewise extracted toxic bodies from normal urines ; and Adduco 



582 THE URINE. 

states that after fatiguing exercise, especially, he could demonstrate 
in the urine a substance which was extremely toxic, and was not iden- 
tical with cholin, as was first supposed. All this work, however, must 
be repeated with great care before the . results obtained can be 
regarded as conclusive. This is also true of the work which has 
been done in various diseases. Some observers have here described 
bodies which they regard as specific toxins. Griffith thus reports 
the presence of a specific poison of scarlatina, of measles, mumps, 
etc. Others again have obtained only negative results. 

The only substances belonging to the class of ptomains which have 
thus far been obtained from the urine in amounts sufficient to estab- 
lish their identity are cadaverin and putrescin. They were originally 
discovered by Brieger in putrefying cadavers, and subsequently also 
found in cultures of the bacillus of Asiatic cholera, the Finkler- 
Prior bacillus of cholerina, the bacillus of tetanus, and in the rice- 
water stools of cholera patients. From the urine cadaverin, putrescin, 
and a third diamin isomeric with cadaverin, and which has been 
regarded as saprin or neuridin, were first obtained by Baumann and 
v. Udranszky in a case of cystinuria, and it appears that dia- 
minuria occurs only in association with this disease. All attempts 
to isolate diamins from the urine under other pathological conditions 
at least have given rise to negative results. Whether or not diamin- 
uria is invariably associated with cystinuria is, however, an open 
question. Putrescin has thus far been found in only three cases, 
viz., in the first case of Baumann and v. Udranszky, in Bodtker's 
case, and in a recent, as yet unpublished, case by Garrod. Brieger, 
Stadthagen, Leo, Garrod, Lewis, and I have succeeded in isolating 
cadaverin from such urines. Others have been less successful, and 
the theory which was announced shortly after Baumann's discovery, 
and quite generally accepted, namely, that the formation of the 
diamins in question is in some manner responsible for the appear- 
ance of cystin in the urine, was certainly premature. This is even 
more true of the inference drawn from this supposed association, 
viz., that cystinuria is a specific infectious disease of the intestinal 
canal. This conclusion was based upon the belief that diamins are 
formed from albuminous material only in the presence of certain 
bacteria. I have shown, however, that this is not necessarily the 
case, and that putrescin at least may be formed in the absence 
of micro-organisms. Further investigation will show whether 
or not cystinuria is invariably accompanied by diaminuria. Per- 
sonally I incline to the belief that this is the case; but I have 
also shown that while cystinuria and diaminuria may coexist, this 
is not always so, and that the two conditions may alternate, and 
that the one may temporarily disappear while the other continues. 
Like Moreigne, I have been led to the conclusion that diaminuria is 
a metabolic anomaly analogous to diabetes and gout, and that both 



PTOMAINS. 583 

diamiuuria and cystinuria are the expression of a marked impairment 
of the normal oxidation-processes of the body. 

The amount of diamins which may be met with in the urine of 
cystinuric patients is extremely variable. In one case I was able 
to isolate as much as 1.6 grammes of the benzoylated cadaverin from 
the collected urine of twenty -four hours. 1 On other days traces 
only were present, and at times, as I have already stated, no diamins 
at all could be found. A few observers who have investigated this 
question, state that they were unable to find even traces of diamins 
in their cases ; but as single examinations only were made, their 
conclusion that diamiuuria does not always accompany cystinuria is 
scarcely justifiable. When single negative results are obtained, the 
examination should be repeated at frequent intervals or larger quan- 
tities of urine employed. In general, I should advise those who 
wish to investigate the question of ptomainuria to experiment with 
large quantities of urine only, as some of the bodies belonging to 
this order exhibit a degree of toxicity which is out of all proportion 
to the amount present. Where specific alkaloids are to be sought 
for, it is scarcely worth while to use less than 100 or 200 liters of 
urine, and even with such amoimts the results are frequently disap- 
pointing. In cases of cystinuria much smaller quantities will 
usually suffice, and an initial experiment may be made with the 
collected urine of twenty-four hours. 

Isolation of Diamins. — Method of Baumann and v. Udranszky. — 
The collected urine of at least twenty-four hours is shaken with a 
10 per cent, solution of sodium hydrate and benzoyl chloride in the 
proportion of 1500 : 200 : 25 until the odor of the benzoyl chloride 
has entirely disappeared. The resulting precipitate contains phos- 
phates, the benzoyl compounds of the normal carbohydrates of the 
urine, and a portion of the benzoylated diamins. These are filtered 
off with the aid of a suction-pump and digested with alcohol. The 
filtered alcoholic extract is concentrated to a small volume and 
poured into about 30 times its amount of water. Upon standing for 
from twelve to forty-eight hours the benzoylated diamins separate out 
in the milky fluid in the form of a more or less voluminous sediment 
composed of fine, intensely white crystals. In order to remove 
the benzoylated carbohydrates likewise present, the precipitate is 
redissolved in alcohol, the solution concentrated to a small volume, 
and diluted with water as described. This process is repeated several 
times. The resulting crystals, if both diamins are present, will lose 
their water of crystallization at 120° C. and melt at 140° C. 

A smaller portion of the benzoyl diamins remains in the first fil- 
trate. In order to recover this, the filtrate is acidified with sulphuric 
acid and extracted with ether. The ethereal residue, before congeal- 

1 In the case of Dr. Lewis, which was examined in my laboratory, 0.3 gramme only 
could be obtained from 12,000 c.c. 



584 THE URINE. 

ing, is placed in as much of a 12 per cent, solution of sodium 
hydrate as is required for its neutralization, when from 3 to 4 times 
the volume of the same solution is added. This mixture is placed 
in the cold, when long needles and platelets separate out, which 
consist of the sodium compound of benzoyl cystin and the benzoy- 
lated diamins. The sediment is filtered off and placed in cold water, 
in which the sodium-benzoyl cystin dissolves, while the benzoylated 
diamins remain undissolved. 

Iu order to separate the putrescin from the cadaverin, the crystals 
are dissolved in a little warm alcohol and treated with 20 times the 
volume of ether. Benzoyl-putrescin is thus thrown down, and may 
be recognized by its melting-point, viz., 175°-176° C, while the 
ethereal residue contains the benzoyl-cadaverin, which melts at from 
129° to 130° C. 

The diamins may then be separated from the benzoyl radicle by 
heating the crystals on a water-bath with a mixture of equal parts 
of alcohol and concentrated hydrochloric acid until a specimen is 
entirely dissolved by sodium hydrate. The separation is complete 
after from twenty-four to forty-eight hours, according to the amount 
present. The solution is then diluted with water, when the benzoic 
acid, which has been formed, separates out and is filtered off. After 
extracting with ether, in order to remove any benzoic acid still 
remaining, the filtrate is evaporated to dryness. A crystalline mass 
remains, which is easily soluble in water but with difficulty in 
alcohol. This consists of putrescin and cadaverin hydrochlorates, 
from which the various double salts with platinum, silver, mercury, 
etc., can be readily obtained. The platinum salt of cadaverin is 
formed by adding an alcoholic solution of platinum chloride to a 
solution of the hydrochlorate in alcohol ; it occurs as a voluminous 
yellow crystalline mass, which can be purified by recrystallization 
from hot water. When this salt is decomposed by hydrogen sulphide 
the hydrochlorate again results, from which the free base is obtained 
by distillation with caustic potash. During this distillation water 
passes over at first ; and above 160° C. a colorless oil appears, the 
boiling-point of which is about 173° C. This constitutes the free 
base, which may be identified by its sperm-like odor and the avidity 
with which it attracts carbon dioxide from the air to form a carbo- 
nate. 

Literature.— Stadthagen, "Ueber d. Harngift," Zeit. f. klin. Med., 1889, vol.xv. 
p. 383. Bouchard. Compt. rend. Soc. de Biol., 1884 ; and Compt. rend, de 1' Acad, des 
Sci., vol. cii. p. 1127. Lepine et Aubert, Ibid., vol. ci. p. 90. Adduco, Arch. ital. 
d. Biol., vol. ix. p. 203, and x. p. 1. 

Diaminuria : v. Udranszky u. Baumann, Zeit. f. physiol. Chem., 1889, vol. xiii. p. 
562. Stadthagen u. Brieger, Berlin, klin. Woch., 1889, vol. xxvi. p. 344. Bodtker, 
Norsk. Mag. f. Laegevidensk., 1892, vol. vii. p. 1220. Moreigne, Arch.de Med. exper. 
et d'Anat. path., 1899, p. 254. Simon, Am. Jour. Med. Sci., 1900, vol. cxix. p. 39. 
Garrod and Cammidge, Jour. Path, and Bact., Feb., 1900. 



MICROSCOPICAL EXAMINATION OF THE URINE. 585 

KRYOSCOPIC EXAMINATION OF THE URINE. 

The kryoscopic examination of the mixed urine does not furnish 
as valuable information as the corresponding examination of the 
blood. This is largely owing to the fact that the normal variations 
in the freezing-point of the urine are much more extensive — i. e., 
between — 0.9° and — 2° C. In the determination of renal insuffi- 
ciency, however, where specimens from each kidney separately are 
available, or at least one specimen for one kidney together w T ith a 
mixed specimen from the same patient, the method furnishes very 
satisfactory results ; it indicates the location of the disease even 
more definitely than a quantitative estimation of urea, tests of specific 
gravity, and the other usual tests of the urine. Especially interest- 
ing are the results which are obtained in cases of unilateral disease 
of the kidneys in which the other organ is functioning normally ; 
kryoscopic examination of the blood will then furnish normal values 
as there is normal elimination, while a separate examination of the 
urine from the two sides reveals the diseased kidney. A value 
of A higher than — 0.9° C. is abnormal. 

The examination is conducted as described in the case of the 
blood. 

Literature. — See page 163. 

MICROSCOPICAL EXAMINATION OF THE URINE. 

Sediments. 

In the chapter treating of the general physical characteristics of 
the urine it was stated that, on standing, every urine gradually be- 
comes cloudy owing to development of the so-called nubecula. This 
was shown to consist of a few mucous corpuscles, a small number 
of pavement epithelial cells derived from the urinary and genital 
passages, and under certain conditions of a few crystals of uric acid, 
of calcium oxalate, or of both. It was further pointed out that 
owing to a diminution in the acidity of the urine on standing, in 
consequence of an interaction between the neutral sodium urate and 
the acid sodium phosphate, a sediment is thrown down which con- 
sists of acid sodium urate, and at times of free uric acid (see Reac- 
tion). Should the reaction of the urine be alkaline, however, when 
freshly voided, a condition which may occur physiologically, when 
it is dependent upon the ingestion of large quantities of vegetables 
rich in organic salts of the alkalies, but which may also be due to 
ammoniacal decomposition, those constituents of the urine which are 
held in solution merely in consequence of the presence of acid sodium 
phosphate are also thrown down. In that case the sediment consists 
essentially of calcium, magnesium, and ammonium salts. Crystals of 
ammonio-magnesium phosphate, it is true, may also be observed in 



586 THE URINE. 

alkaline urines of the first variety, but they are then almost always 
due to an increased elimination of ammonia, and hence are rarely 
observed under physiological conditions. 

Normally calcium is found only in combination with phosphoric 
acid and carbonic acid. Of the three possible calcium salts of phos- 
phoric acid — i. e., Ca 3 (P0 4 ) 2 , CaHP0 4 , and Ca(H 2 P0 4 ) 2 — only the 
first two are found in an alkaline urine, but they may also be observed 
in specimens which are either neutral or but faintly acid. The acid 
calcium phosphate, Ca(H 2 P0 4 ) 2 , is seen but rarely in sediments, and 
its occurrence always presupposes the existence of a high degree of 
acidity ; it is precipitated together with uric acid and under similar 
conditions. Calcium carbonate, CaC0 3 , is seen only in neutral or 
alkaline urines. As soon as ammoniacal fermentation has begun, 
ammonium salts are, of course, formed, viz., ammonium urate and 
ammonio-magnesium phosphate. 

The following table shows the various mineral constituents usually 
observed in sediments, the reaction of the urine being in every case 
the all-important factor : 
Reaction acid: 

Uric acid. 

Sodium urate. 

Calcium oxalate. 

Primary calcium phosphate. 

Ammonio-magnesium phosphate. 
Reaction alkaline (referable to fixed alkalies) : 

Secondary calcium phosphate. 

Tricalcium phosphate. 

Calcium carbonate. 

Ammonio-magnesium phosphate. 
Reaction alkaline (referable to ammonia) : 

Ammonium urate. 

Ammonio-magnesium phosphate. 

Tricalcium phosphate. 

Calcium carbonate. 
In pathological conditions still other unorganized substances may 
be observed, viz., cystin, xanthin, hippuric acid, indigo, urorubin, 
bilirubin, hsematoidin, magnesium phosphate, calcium sulphate, 
cholesterin, leucin, tyrosin, fats, soaps of magnesium and calcium, 
etc. Of these, cystin, xanthin, hippuric acid, tyrosin, calcium sul- 
phate, bilirubin, hsematoidin, magnesium phosphate, leucin, and the 
soaps of magnesium and calcium occur principally in acid urines, 
while indigo, urorubin, and cholesterin are usually only found in 
alkaline specimens. Before considering these various constituents 
in detail, a few words regarding sediments in general and the 
method to be followed in their microscopical examination may not 
be out of place. 



MICROSCOPICAL EXAMINATION OF THE URINE. 587 

An idea of the nature of a deposit may often be formed by simple 
inspection, especially if the reaction of the urine is known. 

A crystalline sediment, presenting a brick-red color and appear- 
ing to the naked eye like cayenne pepper, is usually referable to uric 
acid. On the other hand, a deep-red amorphous deposit occurring 
in an acid urine consists essentially of urates, the color in this case, 
as in the former, being due to uroerythrin. Further proof is hardly 
required. Should doubt be felt, however, it will only be necessary 
to heat the urine, when the deposit will dissolve. A white floccu- 
lent sediment in an alkaline urine is usually referable to a mixture 
of phosphates and carbonates, and will dissolve without difficulty 
upon the addition of acetic acid, but remains unaffected by heat. 

A sediment consisting of pus, and occurring in alkaline urines, is 
frequently mistaken for a phosphatic deposit by the beginner. Aside 
from a microscopical examination, this question may be settled by 
the addition of a small piece of caustic soda and stirring, when in 
the presence of pus the liquid becomes mucilaginous and ropy. If 
much pus is present, a tough, jelly-like mass will be formed, which 
escapes from the vessel en masse when the urine is poured out. 
Such a sediment, moreover, does not disappear upon the addition 
of an acid, and is rendered still more dense upon the application of 
heat. 

Blood when present beyond traces may also be recognized. 

As a general rule, the non-organized elements of a sediment are 
of little clinical interest. 

Students are frequently in the habit of diagnosing an excessive, 
normal, or subnormal elimination of one or another urinary con- 
stituent from the result of a microscopical examination. This is 
unwarrantable, and it should always be remembered that no con- 
clusions whatsoever can be drawn in this manner as to the 
amount actually eliminated. Nothing would be more erroneous 
than to infer an excessive excretion, not to speak of an exces- 
sive production, of uric acid or of oxalic acid from the fact that 
crystals of these substances are seen in large numbers under the 
microscope. Again and again cases are observed in which an ex- 
cessive elimination of uric acid, oxalic acid, or phosphates is diag- 
nosed by mere inspection, and in which a careful chemical analysis 
shows not only no increase, but even a diminution of the normal 
quantity. 

A urine which is turbid when passed may be examined micro- 
scopically at once. As a rule, however, it is necessary to wait until 
a sediment has formed. . To this end, the urine should be kept in 
a clean and well-stoppered bottle. A small amount of chloroform 
is added if necessary, and will preserve the specimen almost in- 
definitely. A few drops of the sediment are then removed by 
means of a clean pipette, carried down to the sediment, with the 



588 THE URINE. 

distal end tightly closed by the finger, care being taken not 
to allow the urine to rush into the tube by suddenly releasing 
the pressure, but withdrawing an amount just sufficient for an 
examination. This is then spread over a clean slide that has 
been moistened by the breath, when the specimen may be exam^ 
ined at once. Covering the specimen with a slip is not only unnec- 
essary, but even undesirable. A low power of the microscope should 
always be employed, and the high power only used to study details 
of structure. 

If a centrifugal machine is available, it is, of course, not necessary 
to let the urine stand until a sediment has formed. An amount 
sufficient for a microscopical examination can then be obtained in a 
few minutes. 

Non-organized Sediments. 

Sediments occurring in Acid Urines. — Uric Acid. — The form 
which uric acid crystals may present in a deposit varies greatly, the 
most common being the so-called" whetstone-form shown in Fig. 107. 

Fig. 117. 




Colorless crystals of uric acid. 

The crystals may occur singly or arranged in groups. Accidental 
impurities, such as threads or hairs, are at times covered with such 
crystals, forming long cylinders. Very frequently uric acid crystal- 
lizes in the form of large rosettes composed of drawn-out whetstone- 
crystals, presenting a deep-red color, referable to uroerythrin, when 
they are often visible to the naked eye, and form the well-known 
brick-dust sediment. While it is generally stated that uric acid 
crystals can always be recognized by their color, which may vary 
from a light yellow to a dark brown, this is, in my experience, not 
the case. I have often seen uric acid sediments in which the 
crystals formed small rhombic plates with rounded edges, and 
were absolutely devoid of coloring-matter, so far as a microscopical 
examination could show (Fig. 117). Uric acid " dumb-bells " are 



MICROSCOPICAL EXAMINATION OF THE URINE. 589 

also at times observed, and may be mistaken for calcium oxalate. 
Hexagonal plates of uric acid have been similarly confounded Avith 
cystin. 

A uric acid sediment may be observed in cases in which an in- 
creased excretion of uric acid occurs ; but it should be remembered 
that, as a rule, it is not permissible to infer an increased production 
or elimination from the presence of an abundant deposit of this sub- 
stance alone. Brick-dust sediments are frequently observed during 
cold weather ; but it would be erroneous to infer an increased elimi- 
nation from sucl> an occurrence, as the phenomenon is owing to 
the fact that uric acid is less soluble in cold than in warm water. 
During the summer months, for the same reason, a deposit of uric 
acid is less frequently observed, although an increased amount may 
nevertheless be present, being held in solution owing to the higher 
temperature. The more concentrated the urine and the more uric 
acid it contains, the more readily will such a deposit form. It is 
hence noted after profuse perspiration, following severe muscular 
exercise, in acute rheumatism with copious diaphoresis, in acute 
gastritis and enteritis associated with copious vomiting or diarrhoea, 
during the crisis of pneumonia (particularly if accompanied ' by 
much sweating), etc. In all these conditions, however, an increased 
elimination of uric acid does not necessarily take place, the all- 
important factors being the reaction of the urine, its degree of con- 
centration, and the surrounding temperature. 

Should formed concretions of uric acid — i. e., uric acid gravel — 
be found in the urine, a direct indication is afforded to diminish the 
acidity of the urine and to increase the amount of water, so as to 
guard against the formation of renal or vesical calculus. 

Chemically, the nature of a uric acid sediment may be recognized 
by the fact that the crystals dissolve upon the addition of sodium 
hydrate, and reappear in the rhombic form upon acidifying with 
hydrochloric acid. When heated with dilute nitric acid the beauti- 
ful red color of ammonium purpurate is obtained upon the subsequent 
addition of ammonia (murexid test), as described elsewhere (see page 
449). 

Amorphous Urates. — Sodium and potassium urate frequently, and 
especially in fevers, form sediments of such density that upon 
microscopical examination it is almost impossible to discern anything 
but innumerable amorphous granules scattered over the entire field 
and obscuring all other elements that may be present. Cells or 
casts will frequently be seen studded with these granules. In such 
cases it is best to heat the urine to a temperature of 50° C, and to 
filter it as rapidly as possible while hot, the contents of the filter 
being subsequently used for a microscopical examination. 

Urate sediments are always colored, the tint varying from a dirty 
brown to a bright salmon-red, owing to the presence of uroerythrin. 



590 THE URINE. 

Difficulties can hence never arise in determining the nature of the 
sediment, as a colored deposit appearing in an acid urine which dis- 
solves upon the application of heat cannot be due to anything but 
urates. If a drop of the sediment, moreover, is treated upon a 
slide with a drop of hydrochloric acid, characteristic whetstone- 
crystals of uric acid separate out, but the greater portion appears in 
the form of rhombic platelets. 

Calcium Oxalate. — This substance generally appears in urinary 
sediments in the form of colorless, highly refractive octahedra (Fig. 
118), which vary greatly in size; some appear as mere specks 
under even a comparatively high power, while others may attain 
the dimensions of a large leucocyte. Frequently one axis is 
longer than the other. From the fact that their diagonal planes 
are highly refractive, apparently dividing the superficial plane 
into four triangles, they have been compared to envelopes, and it 
is this envelope-form of the crystals which is especially character- 
istic. In the same specimen of urine so-called dumb-bell forms may 
be seen, which appear to be made up of two bundles of needle-like 
crystals united in the form of the figure 8. These, according to 
Beale, originate in the uriniferous tubules, and are frequently found 
adherent to or imbedded in tube-casts. Other forms may also be 
seen, and are shown in the accompanying figure. 

While the envelope crystals are highly characteristic and can 
hardly be mistaken for any other substance, the student may at times 
confound them with crystals of amnion io-magnesium phosphate. 
This error may be avoided if it is remembered that the calcium oxa- 
late crystals are usually not so large as those of the magnesium salt, 
and that the latter dissolve upon the addition of acetic acid, in which 
calcium oxalate is insoluble. The distinction from uric acid, if we 
are dealing with the dumb-bell form, cannot always be made by 




Less common forms of calcium oxalate crystals. (Finlayson.) 

mere inspection. A drop of caustic soda should be added, which 
will dissolve the crystals if these are uric acid, while calcium oxalate 
remains unchanged. 



MICROSCOPICAL EXAMINATION OF THE URINE. 



591 



It has been pointed out that under strictly normal conditions a 
few isolated crystals of calcium oxalate may be found in the primi- 
tive nubecula, so that their presence in urinary sediments cannot be 
regarded as pathological. After the ingestion of certain vegetables 
and fruits, notably rhubarb, garlic, asparagus, and oranges, or follow- 
ing the continued administration of sodium bicarbonate or the salts 
of vegetable acids, calcium oxalate crystals may be observed in large 
numbers ; so also in certain diseases, such as diabetes mellitus, catar- 
rhal jaundice, phthisis, emphysema, etc. 

As in the case of uric acid, no inference as to the quantity 
eliminated can be drawn from a microscopical examination of the 
sediment. The frequent occurrence of abundant sediments of this 
substance may, however, generally be regarded as abnormal, pro- 
viding that such an occurrence cannot be explained by the nature 
of the diet. It is very suggestive to note the frequency with 
which such sediments are observed in cases of neurasthenia, asso- 
ciated with a mild degree of albuminuria, as also in various di- 
gestive neuroses. Finally, as with uric acid, the possibility of the 
formation of renal calculi should be borne in mind whenever abun- 
dant sediments of calcium oxalate are encountered upon frequent 
examination. 

Ammonio-magnesium phosphate, usually spoken of as triple phos- 
phate, crystallizes in large prismatic crystals of the rhombic system ; 

Fro. 119. 




Various forms of triple phosphates. (Finlayson.) 

it is most abundantly observed in alkaline urines, but may also occur 
in feebly acid specimens. Of the various forms which may occur, 
that resembling the lid of a German coffin is the most characteristic 
(Fig. 119). At times these crystals attain considerable size ; very 
small specimens, however, also occur which may be mistaken for 
oxalate of calcium, but from these they are readily distinguished 
by the ease with which they dissolve in acetic acid, as has been 
pointed out. 

Here, as elsewhere, it should be remembered that no conclusions 



592 



THE URINE. 



as to the amount actually eliminated can be drawn from a micro- 
scopical examination, and the diagnosis " phosphaturia " should be 
based only upon the results of a quantitative analysis. 



Fig. 120. 




Crystalline phosphates. (Finlayson.) 

The continued elimination of a turbid urine, the turbidity of which 
is referable to phosphates, is notably observed in neurasthenic indi- 
viduals with a predominance of cerebral symptoms. Very curiously, 
the phosphaturia is not influenced by diet. 

Monocalcium phosphate crystals are rarely seen, and only in speci- 
mens presenting a highly acid reaction, when uric acid crystals are 
also frequently observed in large numbers. I have seen only a few 
cases of this kind, occurring in patients the subjects of functional 
albuminuria. The urine was highly acid, in one case of a specific 
gravity of 1.036, and on standing deposited a sediment which con- 
sisted largely of monocalcium phosphate crystals (Fig. 121), with a 
considerable number of uric acid crystals, from which they are 



Fig. 121. 




Monocalcium phosphate crystals. 

readily distinguished by the absence of pigment and their solubility 
in acetic acid. 



MICROSCOPICAL EXAMINATION OF THE URINE. 593 

Neutral Calcium Phosphate. — These crystals may be found in alka- 
line, neutral, and feebly acid urines. They are at times of large 
size, but more commonly acicular, occurring either singly or united 
in a star-like manner (Fig. 120). They are colorless, readily solu- 
ble in acetic acid, and insoluble in warm water, so that they can be 
easily distinguished from uric acid. 

Basic magnesium phosphate crystals occurring in the form of large, 
highly refractive plates (Fig. 122), are at times seen in alkaline, 
neutral, or faintly acid and highly concentrated urines. They are 
readily recognized by treating a drop of the sediment upon a slide 
with a drop of ammonium carbonate solution (1 : 4), when the crys- 
tals become opaque and their edges assume an eroded aspect. In 
acetic acid they dissolve with ease and may then be reprecipitated 
by means of sodium carbonate. 1 

Hippuric acid crystals have been observed, although rarely, in uri- 
nary sediments, in acute febrile diseases, diabetes, and chorea ; while 
their occurrence following the ingestion of large amounts of prunes, 
mulberries, blueberries, or the administration of benzoic acid and 
salicylic acid, is more common. 

Fig. 122. 




^O 1 



Basic magnesium phosphate crystals, (v. Jaksch.) 

Hippuric acid occurs in the form of fine needles or rhombic prisms 
and columns, the ends of which terminate in two or four planes, at 
times resembling the crystals of ammonio-magnesium phosphate and 
of uric acid. From the former they may be readily distinguished by 
their insolubility in hydrochloric acid, and from the latter by the 
fact that they do not give the murexid reaction when treated with 
nitric acid and ammonia (see page 449). In the case of urines rich 
in hippuric acid in which the substance does not appear in the sedi- 
ment, it is well to add a small amount of hydrochloric acid, when 
the crystals will gradually separate out. Their presence does not 
appear to possess any clinical significance. 

Calcium sulphate, in the form of long colorless needles or elon- 
gated prismatic tablets (Fig. 123), has been observed in urinary 
sediments in only two cases. In both the urine, especially on 
standing, deposited a milky-looking sediment, the reaction being 

1 Stein, Arch. f. kliu. Med., 1876, vol. xviii. p. 207. 
38 



594 



THE URINE. 



strongly acid. It may be recognized by its insolubility in acids and 



ammonia. 



Fig. 123. 




Calcium sulphate crystals, (v. Jaksch.) 



Cystin (C 6 H 12 4 S 2 ) is rarely seen in urinary sediments. It occurs 
in the form of colorless hexagonal platelets, which are very charac- 
teristic (Fig. 124). The crystals are soluble in ammonia and hydro- 
chloric acid, and insoluble in acetic acid, water, alcohol, and ether. 



Fig. 124. 




Crystals of cystin spontaneously voided with urine. (Roberts.) 

They can thus be readily distinguished from certain forms of uric 
acid, with which they might possibly be confounded at first sight. 
When heated upon platinum foil they burn with a bluish-green 
flame without melting. 

Cystin-containmg urines may be of normal appearance, but they 
often present a peculiar greenish-yellow color. Their reaction is 
mostly neutral or alkaline. Upon exposure to the air a marked odor 

1 v. Jaksch, Zeit. f. klin. Med., 1892, vol. xxii. p. 554. 



MICROSCOPICAL EXAMINATION OF THE URINE. 595 

of hydrogen sulphide develops, owing to decomposition of the cystin ; 
but at times urines are met with in which a distinct odor of hydrogen 
sulphide is noticeable, although crystals of cystin are not seen in the 
sediment. It may then be demonstrated by strongly acidifying the 
urine with acetic acid or by allowing it to undergo ammoniaeal decom- 
position. In either case cystin crystals will separate out on standing. 
It should be remembered, however, that not all urines in which 
hydrogen sulphide is formed contain cystin (see Hydrothionuria). 

The amount of cystin which may be found in urinary sediments 
is variable. Sometimes a few centigrammes only are obtained, while 
at others from 0.5 to 1 gramme may be recovered. As is the case 
with the other non-organized constituents of sediments, however, the 
amount deposited does not necessarily indicate the total amount 
present. Where a quantitative estimation of cystin is to be made, 
it is best to filter off that which is deposited and to estimate the 
amount of neutral sulphur in the filtered urine. An increase beyond 
the normal may be referred to the cystin remaining in solution (see 
Neutral Sulphur). 

Clinical interest in connection with cystinuria centres in the fre- 
quent association of cystin sediments with cystin gravel or calculi ; 
but it is curious to note that the cystinuria, notwithstanding the 
removal of the calculus, may persist for years without giving rise to 
symptoms denoting the existence of a pathological process. 

Very remarkable is the not uncommon occurrence of cystinuria in 
families. Cases of transient cystinuria likewise occur, and it is 
hence scarcely admissible to speak of a " cured " cystinuria when 
the condition disappears under treatment. 

Of the origin of the condition little is known. It has been sup- 
posed that the appearance of cystin in the urine is in some manner 
connected with the formation of certain diamins in the intestinal 
canal. I have pointed out, however, that in all probability the for- 
mation of cystin and diamins takes place in the tissues of the body, 
and that the appearance of both is the expression of a definite meta- 
bolic anomaly rather than of a specific infection (see page 583). 

Literature. — C. E. Simon, " Cystinuria and its Eelation to Diaminuria," Am. 
Jour. Med. Sci., 1900, vol. cxix. p. 39. See also the literature on page 584. 

Leucin and tyrosin belong to the group of amido-acids, and are 
represented by the formulae C 6 H 13 N0 2 and C 9 H n 3 . They are never 
found in urinary sediments under normal conditions, while traces of 
both substances may be present in solution. Larger amounts are 
notably found in acute yellow atrophy, of which disease their presence 
in sediments is almost pathognomonic. In acute phosphorus poison- 
ing leucin and tyrosin are usually not found. The fact that urea 
may be altogether absent from the urine in acute yellow atrophy or 
present in greatly diminished amount has been previously referred 



596 



THE URINE. 



to (see Urea, page 421), and the elimination of leucin and tyrosin 
in its stead, as it were, has been regarded not only as indicating the 
probable origin of urea from amido-acids, but also the formation of 
urea, to a large extent at least, in the liver. The albuminous origin 
of these substances has also been noted (see Urea). 

Traces of leucin and tyrosin are said to be constantly present in 
cases of cirrhosis and carcinoma of the liver, in cholelithiasis, catar- 
rhal jaundice, Weil's disease, nephritis, cystitis, gout, bronchitis, 
tuberculosis, typhoid fever, hysteria, erysipelas, glucosuria, etc. In 
connection with cystinuria, the elimination of tyrosin has also been 
observed, but in two cases which I examined in this direction I 
obtained negative results. In diabetic urines both are supposedly 
absent. 

As leucin is hardly ever found in the sediment, and tyrosin only 
when present in large quantities, the urine in every case should first 
be concentrated upon a water-bath and examined on cooling. At 
times, however, when these substances are present in only very small 
quantities, this procedure may not lead to the desired end, and in 
doubtful cases the following method should be employed : 

The total amount of urine voided in tweuty-four hours is pre- 
cipitated with basic lead acetate and filtered, when the filtrate, from 
which the excess of lead has been removed by means of hydrogen 
sulphide, is evaporated to as small a volume as possible, and is set 
aside for crystallization. The residue thus obtained is then examined 
with the microscope ; if crystals are detected which answer the 
description of tyrosin and leucin, they should be subjected to further 
chemical tests. 

Fig. 125. 




Tyrosin crystals. (Charles.) 



Ulrich advises to evaporate the urine to dryness and to heat the 
residue gently while the vessel is covered w T ith a plate of glass or a 
funnel. The tyrosin is then said to sublime, and is deposited on the 
cool glass in crystalline form, the crystals giving the characteristic 
reactions. 

Tyrosin crystallizes in the form of very fine needles (Fig. 125), 



MICROSCOPICAL EXAMINATION OF THE URINE. 



597 



which are usually grouped in sheaves or bundles crossing each other 
at various angles. They are insoluble in acetic acid, but soluble in 
ammonia and hydrochloric acid. 

Leucin (Fig. 126) occurs in the form of spherules of variable size, 
which closely resemble globules of fat, but may be distinguished from 
these by their insolubility in ether. In the urine they present a 
more or less pronounced brownish color, and upon close examination 
concentric striations as well as very fine radiating lines can at times 
be made out, which are especially characteristic. 

If crystals resembling tyrosin and leucin are found, the following 
tests should be made : 

Tests for Tyrosin. — The sediment is filtered off, washed with 
water and dissolved in ammonia to which a little ammonium car- 
bonate has been added. The solution is allowed to evaporate, when 
the tyrosin remains behind. 

Piria's Test 1 — A bit of the tyrosin is moistened on a watch-crys- 
tal with a few drops of concentrated sulphuric acid, covered, and 
set aside for half an hoar. It is then diluted with water, heated, 
and while hot saturated with calcium carbonate and the solution 
filtered. The filtrate is colorless, but when heated with a few drops 
of a very dilute solution of ferric chloride, which must be. free from 
hydrochloric acid, it assumes a violet tint (v. Jaksch). 

Fig. 126. 




Crystals of leucin (different forms). (Crystals of kreatinin-zinc chloride resemble the 
leucin crystals depicted at a.) The crystals figured to the right consist of comparatively 
impure leucin. (Charles.) 

Hoffmann's Test 2 — A small amount of tyrosin is dissolved in hot 
water and treated, while hot, with mercuric nitrate and potassium 
nitrite. The solution assumes a beautiful dark-red color and yields 
a voluminous red precipitate. 

Tests for Leucin. — Scherer's Test 3 — To test for leucin, this is 
separated from tyrosin by the addition of a little alcohol (see below). 
The alcohol is allowed to evaporate, and a portion of the residue 



1 Piria, Liebig's Annal., 1852, vol. lxxxii. p. 251. 

2 Hoffmann, Ibid., 1857, vol. lxxxvii. p. 124. 

3 Scherer, Jour. f. prak. Chem., 1887, vol. lxxix. p. 410. 



598 THE URINE. 

treated upon platinum foil with nitric acid, when a colorless residue 
is obtained which, upon the application of heat and the addition of a 
few drops of a solution of sodium hydrate, forms a droplet of an 
oily fluid which does not adhere to the platinum. 

Hofmeister' s Test 41 — A small amount of leucin dissolved in water 
causes a deposit of metallic mercury when heated with mercurous 
nitrate. 

In order to separate the leucin from the tyrosin, the sediment is 
treated with a small amount of alcohol, in which leucin is more 
readily soluble than tyrosin. 

Litekattjre. — Frerichs, Wien. med. Woch., 1854, vol. iv. p. 465. Schultzen u. 
Riess, Charite Annal., vol. xv. Pouchet, Maly's Jahresber., 1880, vol. x. p. 248. Irsai, 
Ibid., 1885, vol. xiv. p. 451. Prus, Ibid., 1888J vol. xvii. p. 345. Frankel, Berlin, klin. 
Wocb., 1878, vol. xv. p. 265. 

Xanthin crystals (Fig. 127) are very rarely observed in urinary 
sediments, and, so far as I have been able to ascertain, the case 
observed by Bence Jones 1 is the only one on record. Care should 
be had not to confound certain forms of uric acid with xanthin, 
and I well remember an instance in which crystals were observed 

Fig. 127. 






j> 



o 



a, Crystals of xanthin (Salkowskx) ; b, Crystals of cystin (Robin). 



identical in appearance with those here pictured, but which upon 
chemical examination proved to be uric acid. The necessity of disre- 
garding the statement generally made that uric acid crystals found in 
urinary sediments are invariably colored cannot be insisted upon too 
strongly. It has been stated elsewhere that colorless uric acid 
crystals may be encountered, and in the case just cited such were 
observed. 

Clinically, xanthin sediments are of interest only in so far as this 
substance may give rise to the formation of calculi ; in the case 
observed by Bence Jones attacks of renal colic had occurred several 
years previously. 

Soaps of Lime and Magnesia. — v. Jaksch has pointed out that 
in various diseases crystals may be found which " closely " resemble 

1 Hofmeister, Liebig's Annal., 1877, vol. cxxxix. p. 6. 

2 Bence Jones, Chem. Centralbl., 1868, vol. xiii. 



MICROSCOPICAL EXAMINATION OF THE URINE. 



599 



tyrosin in appearance, and pictures such crystals (Fig. 128), which 
from their behavior toward reagents he is inclined to regard as cal- 
cium and magnesium salts of certain higher fatty acids. 



Fig. 128. 




Lime and magnesium soaps, (v. Jaksch.) 

Should doubt arise, the question may be readily decided by a 
chemical examination (see tests for tyrosin and fatty acids). 

Bilirubin crystals in the form of yellow or ruby-red rhombic plates 
or needles, as well as amorphous granules, have been seen in the 
urine in rare cases, but are of no special interest. They are easily 
soluble in alkalies and chloroform, but not in ether. When treated 
upon a slide with a drop of nitric acid a green ring will be seen to 
form around them (Gmelin's reaction). 1 Such crystals have been 
found in icteric urine and in a case of pyelonephritis. 

Haematoidin crystals are likewise only rarely seen. They cannot 
be distinguished from bilirubin, with which, indeed, they are sup- 
posedly identical. 2 They may be found either free or imbedded 
within cells or tube-casts, in cases of scarlatinal nephritis, the 
nephritis of pregnancy, in granular atrophy, amyloid degeneration 
of the kidneys, and in carcinoma of the bladder, of which latter 
condition they have been regarded by some as pathognomonic. 

Fat. — When small, strongly refractive globules of fat, which may 
be readily recognized by their solubility in ether, are observed either 
floating on the urine or held in suspension, it is necessary to ascer- 
tain first of all whether such fat may not be present accidentally, 
owing to the use of a bottle or vessel not absolutely clean, or 
previous catheterization, etc. The diagnosis lipuria should only 
be made when all possible precautions have been taken to insure 



1 Kussmaul, Wurzburger med. Zeit., 1863, vol. iv. p. 64. 
Med., 1879, vol. xiii. p. 115. 



Ebstein, Arch. f. klin. 



Hoppe-Seyler u. Thierfelder, Handb. d. physiol. u. path., chem. Analyse. 



600 THE URINE. 

against the accidental presence of this substance. Every phy^ 
sician who has frequent occasion to examine urines has undoubtedly 
met with instances in which fat-globules were found, and in 
which careful inquiry showed that these were accidentally present. 
True lipuria — i. e., an elimination of fat usually in the form of 
droplets floating on the urine — has been noted in various cachectic 
conditions, in cases of heart-disease, affections of the pancreas 
and liver, in gangrene and pyaemia, in diseases of the bones, 
especially following fractures, in diseases of the joints, etc. Fat 
has also been observed in the urine following the ingestion of 
large amounts of cod-liver oil and inunctions with fats and oils. 

In fatty degeneration of the kidneys, in Bright' s disease, phos- 
phorus poisoning, etc., droplets of fat may be seen in the epithelial 
cells and tube-casts. This, however, does not constitute lipuria. 
The nature of the droplets may be recognized by their solubility in 
ether, benzol, chloroform, carbon disulphide, xylol, etc., and by the 
fact that they are colored black when treated with a 0.5 to 1 per 
cent, solution of osmic acid, and red when a drop of tincture of 
alcanna is added to the specimen. A very convenient method of 
demonstrating the presence of fat is also the following : a few 
cubic centimeters of the urine are mixed with an equal volume of 
96 per cent, alcohol and a concentrated solution of Sudan III. in 
96 per cent, alcohol. The sediment which collects is then ex- 
amined under the microscope ; the excess of stain is removed by 
allowing a few drops of 60 or 70 per cent, alcohol to run under 
the cover-slip and removing it with filter-paper placed at the 
edge of the preparation. The fat-droplets are thus colored 
an intense scarlet red, while granules of albuminous origin are 
unstained. Free fat can, of course, be demonstrated in the same 
manner. 

The largest amounts of fat are observed in chyluria, a condition 
which is usually due to the presence of a specific parasite in the 
blood, viz., the Filaria sanguinis hominis, or more rarely the Distoma 
haBmatobium, which have been described in the chapter on the Blood 
(see also Chyluria). 

Sediments occurring in Alkaline Urines. — Basic Phosphate of 
Calcium and Magnesium. — The most common sediments observed in 
alkaline urines consist of amorphous phosphates of calcium and 
magnesium. They are usually as abundant as the urate sediments 
which have been described, but may be readily distinguished from 
these by the fact that they do not dissolve upon the application of 
heat, but readily, disappear upon the addition of acetic acid, and are 
never colored. In this manner it is also easy to distinguish such a 
sediment from one due to pus, with which it might possibly be con- 
founded at first sight. Upon microscopical examination a drop of 
the sediment will be seen to contain innumerable transparent granules 



MICROSCOPICAL EXAMINATION OF THE URINE. 601 

scattered over the entire field, and closely resembling those of urate 
of sodium and potassium. 

Phosphatic sediments are observed, as mentioned elsewhere, when- 
ever the reaction of the urine is alkaline, whether this be owing to 
the presence of fixed alkali or to ammoniacal fermentation. 

Ammonium urate is observed only in urines which are undergoing 
ammoniacal decomposition. Its presence should always call for a 
careful investigation in order to ascertain whether this has taken 
place after the urine has been voided or before (see Reaction). 

The salt occurs in the form of colored spherical bodies of variable 
size, which are sometimes composed of delicate needles, while at 
others they are amorphous, but may be beset with prismatic spicules. 
They are not easily mistaken for any other substance which may be 
present in urinary sediments (Fig. 129). Ammonium urate is 
characterized, moreover, by its solubility in acetic and hydrochloric 
acids, and by the subsequent separation of rhombic crystals of uric 
acid. 

Magnesium phosphate has been described above (see page 593). 

Ammonio-magnesium Phosphate. — While the well-known coffin-lid 
crystals are commonly seen in feebly acid urines, as pointed out, 
ammonio-magnesium phosphate presents a great variety of forms in 
alkaline urines, and especially in specimens undergoing ammoniacal 
decomposition (see Fig. 120). 




Ammonium urate crystals. 

Calcium carbonate frequently occurs in alkaline urines, and appears 
under the microscope in the form of minute granules, occurring 
singly or arranged in masses ; dumb-bell forms are also seen (Fig. 
130). They may be recognized by the fact that they readily dis- 
solve in acetic acid with the evolution of gas. 

Indigo in the form of delicate blue needles, arranged in a stellate 
manner or in plates, visible only with the microscope, is rarely seem 



602 THE URINE. 

In an amorphous condition, however, it may be met with in almost 
every decomposed urine, occurring in the form of small granules, 

Fig. 130. 




Calcium carbonate crystals. 

and frequently staining the morphological elements that may be 
present a distinct blue. Sediments presenting a bluish-black color 
were noted in the time of Hippocrates already, and have been 
described since by numerous observers, but the nature of the color- 
ing-matter has only been determined within the last fifty years. 
Clinically, the occurrence of indigo in the urine is of interest, as 
renal calculi have been observed which consisted almost entirely of 
this substance. But little is known of the causes which give rise 
to its appearance in the urine, but there can be no doubt that its 
occurrence is referable to the action of certain micro-organisms 
upon urinary indican (see page 559). 1 

Organized Constituents of Urinary Sediments. 

Epithelial Cells (Fig. 131). — Bearing in mind the fact that 
desquamative processes are constantly going on in the epithelial 
lining of the various cavities and channels of the body, one should 
expect to find in every urine representatives of the different forms 
of epithelium occurring in the urinary organs, from the Malpighian 
tufts down to the meatus urinarius. To a certain extent this actu- 
ally happens, and cells apparently derived from the meatus, the 
urethra, bladder, ureters, and pelvis of the kidneys may be met with 
in almost every specimen, although it may at times be difficult to 
refer to their origin the individual cells observed. Bizzozero even 
claims that it is impossible to distinguish between the cells of 
the bladder and those of the meatus and renal pelvis, while as a 
class they may readily be differentiated in most cases from the cells 
of the urethra, the ureters, the prepuce of the male, and the vulva 
and vagina of the female. Cells from the uriniferous tubules of the 
kidneys are seldom seen in normal urines, and when they do occur 
it is impossible to determine their exact origin — i. e., the particular 

1 v. Jaksch, Prag. nied. Woch., 1892, vol. xvii. p. 602. 



MICROSCOPICAL EXAMINATION OF THE URINE. 



603 



portion of the tubule from which they have been detached. Ceils 
presenting the characteristic striated appearance seen in the irregu- 
lar, and to a less evident degree in the convoluted, portions of the 
uriniferous tubules, are never observed in the urine. This fact, as 
well as the usual absence of true glandular cells, remains to be 
explained. It is not improbable that the absence of these cells may 
be referable to a less marked desquamation going on in those parts 
in which the mechanical injury to which the epithelium is subject 
must of necessity be far less severe than in the remaining portions 
of the urinary tract, and particularly in the bladder and urethra. 



Ftg. 131. 




Epithelium from the urinary passages. 
a, Round cells ; b, conical and caudate cells ; c, flat cells. 

As stated elsewhere, the number of epithelial cells occurring in 
urinary sediments under physiological conditions is small, and the 
presence of large numbers may hence always be regarded as abnormal, 
and indicating the existence of a circulatory or inflammatory dis- 
turbance affecting some portion of the urinary tract. 

Were it possible in every case to determine the exact origin of 
the cells, it is evident that information of great value would thus 
be obtained. Unfortunately, this is not always possible, as the 
form of the cells is dependent to a certain extent upon the reac- 
tion of the urine, an alkaline or neutral reaction causing the cells 



604 THE URINE 

to swell and to appear larger and rounder than is the case in acid 
urines. As has been mentioned, the cellular type is practically the 
same, moreover, in the bladder, ureters, and pelvis of the kidneys. 

Definite conclusions should hence be drawn only exceptionally 
from a microscopical examination alone, but there can be no doubt 
that in conjunction with other factors and the clinical history the 
demonstration of a normal or increased number of epithelial cells 
may frequently be of decided value in a differential diagnosis, and 
taking these factors into consideration it may even be possible to 
localize the seat of the lesion. If attention is directed to the struct- 
ure of the individual cell — and this holds good more especially for 
the cells derived from the uriniferous tubules — an idea may at times 
even be formed of the character of the lesion (see below). 

Ultzmann recognizes three forms of epithelial cells which may be 
found in urinary sediments, viz.: 

1. Round cells. 

2. Conical and caudate cells. 

3. Flat cells. 

Round cells are usually derived from the uriniferous tubules and 
the deeper layers of the mucous membrane of the pelvis of the kid- 
neys. In the urine they present a more or less rounded form and 
are provided with a distinct nucleus ; they are not much larger than 
pus-corpuscles. From the latter they are distinguished by the pres- 
ence of a w T ell-defined nucleus, which in pus-cells becomes distinct 
only upon the addition of acetic acid, and is, moreover, polymor- 
phous. Whenever such cells are found adhering to urinary casts, 
which may at times consist entirely of these structures, it is clear 
that they represent the glandular elements proper of the kidneys. 
As similar cells are found in the male urethra, confusion may 
possibly arise. Should albumin, however, be present, the cells are 
probably of renal origin. The presence of such cells in large 
numbers together with pus, in the absence of tube-casts and 
albumin beyond traces, will usually indicate the existence of a 
simple pyelitis, particularly if round cells are found joined in a 
shingle-like manner. Should the pyelitis be associated with a ne- 
phritis, tube-casts and albumin in larger amounts will at the same 
time be present. In such cases it may be impossible to determine 
the origin of the cells, excepting of such that may adhere to casts. 
In simple circulatory disturbances affecting the renal parenchyma 
no special abnormalities can be discovered in the structure of the 
cells, while in cases of fatty degeneration of the kidneys they will 
be seen to contain fatty particles in greater or less abundance, so 
that it may be possible to determine the existence of degenerative 
processes which may be of inflammatory or non-inflammatory origin. 
The same may be said to hold good if the epithelial elements are 
markedly granular and occur in fragments. 



MICROSCOPICAL EXAMINATION OF THE URINE. 605 

Conical and caudate cells are mostly derived from the superficial 
layers of the pelvis of the kidneys, and are hence especially seen in 
cases of pyelitis. Similar cells are also found in the neck of the 
bladder, and may usually be distinguished from those of the pelvis 
by the greater length of their processes. 

Flat cells may come from the ureters, the bladder, the prepuce of 
the male, and the vulva and vagina of the female. These cells pre- 
sent the usual characteristics of squamous epithelium, being large, 
polygonal in form, and provided with a well-defined nucleus ; the 
extra-nuclear protoplasm is only slightly granular. Other more or 
less rounded forms are also seen, which are derived from the deeper 
layers of the mucosa, but may be distinguished from the small 
round cells of the kidneys proper. Irregular or conical cells, often 
provided with one or more protoplasmic processes, likewise come 
from the lower layer of the mucosa of the bladder and ureters. 

While the cells of the bladder may thus be confounded with those 
of the ureters and vagina under the microscope, it is not likely that 
a vaginitis or vulvitis will be mistaken for a cystitis or a ureteritis. 
In doubtful cases specimens of urine should be procured by means 
of the catheter, care being taken to first thoroughly cleanse the vulva. 
The warped appearance so frequently seen in vaginal epithelial cells, 
and the fact that they often and indeed usually appear in masses, 
may further aid in the differential diagnosis. 

It has been pointed out by Peyer that the presence of pavement- 
epithelial cells, together with mucus and leucocytes, in the urine of 
hysterical and anaemic girls may be regarded as indicating an irrita- 
ble condition of the genitals, possibly in consequence of masturba- 
tion. Bearing in mind the moist and sensitive condition of the 
vulva of female masturbators, such a view is plausible. 

A ureteritis, notwithstanding the fact that the ureteral cells 
closely resemble those of the bladder, may be inferred indirectly, 
the presence of squamous cells in abundance pointing to a cystitis, 
a small increase in their number to ureteritis. In conclusion, it 
should be stated that the so-called mucous corpuscles present in 
every urine are young vesical cells. 

From what has been said, it is clear that, with due precautions 
and taking other factors into consideration, the discovery of epi- 
thelial cells in large numbers in urinary sediments may be of decided 
value in diagnosis. 

Literature.— Bizzozero, loc. cit. Eichhorst, Lehrbuch d. physikal. Untersuch. 
inn. Krankheit., 2d ed.. p. 336, Braunschweig. 

Leucocytes. — Leucocytes are encountered in only very small 
numbers in normal urines. A marked increase should, hence, alwavs 
be regarded as indicating the existence of disease somewhere in the 
course of the urinary tract, excepting in females, where their presence 



606 THE URINE. 

may be owing to an admixture of leucorrhoeal discharge. In that 
case the source of the pus will generally be recognized by the simul- 
taneous occurrence of pavement epithelial cells of the vaginal type 
in correspondingly large numbers. In doubtful cases the urine 
should always be obtained with the catheter, care being taken to 
thoroughly cleanse the vulva before the introduction of the instru- 
ment. 

Occasionally the pus is derived from a neighboring abscess that 
has opened into the urinary passages. 

The amount of pus which may be found in urines is most varia- 
ble. On the one hand, deposits several centimeters in height are not 
uncommon, and closely resemble deposits of phosphates in appear- 
ance, for which they are indeed frequently mistaken ; on the other 
hand, it may only be possible to discover the presence of pus by 
means of the microscope, which should be employed in every case. 

The appearance of the pus-corpuscles likewise varies in different 
cases. In acid urines their form is usually well preserved, and in 
feebly alkaline and neutral specimens it may even be possible to 
observe amoeboid movements when the slide is carefully warmed. 
In alkaline urines, however, they usually swell up and become 
opaque, so that it is impossible to discern a nucleus unless they 
are treated with acetic acid. At other times, and particularly when 
pus has remained long in the body, as where an abscess has burst 
into the urinary passages, it may be almost impossible to make out 
a nucleus, and in extreme instances nothing but a mass of granular 
and fatty detritus is left. 

While with a certain amount of experience it is hardly likely that 
a sediment of pus will be mistaken for anything else, such as a 
deposit of phosphates, it should be remembered that if pus is 
exposed to the action of ammonia or an ammonium salt the pus- 
corpuscles become disintegrated. In such cases, as in cystitis, in 
which ammoniacal decomposition of the urine has taken place in the 
bladder, a deposit may be obtained which macroscopically resembles 
mucus, and in which pus-corpuscles may not even be demonstrable 
with the microscope. The sediment then escapes as a gelatinous, 
slippery mass when the urine is poured from one vessel into an- 
other. Recourse must then be had to certain chemical tests, as a 
pyuria might otherwise be overlooked. To this end, the following 
procedure, suggested by Vitali, 1 may be employed : 

The urine, after having been acidified with acetic acid, is filtered, 
and the contents of the filter treated with a few drops of tincture 
of guaiacum which has been kept in the dark, when in the pres- 
ence of pus the filter-paper is colored a deep blue. The reaction is 
supposedly due to the presence in the leucocytes of specific nucleo- 
proteids. 

1 Vitali, Maly's Jahresber., 1890, vol. xviii. p. 326. 



MICROSCOPICAL EXAMINATION OF THE URINE. 607 

A solution of iodo- potassic iodide may be employed in less 
extreme instances. A drop of this solution is added to a drop of 
the sediment upon a slide, when the pus-corpuscles, owing to the 
presence of glycogen, are colored a dark mahogany-brown, while 
epithelial cells, with certain forms of which they might possibly be 
mistaken, assume a light color. 

Donn&s pus-test is based upon the fact that the transformation of 
pus into a gelatinous, mucus-like mass, observed in cases of cystitis, 
owing to the action of ammonium carbonate, may also be artificially 
produced by the addition of a small piece of caustic soda and stir- 
ring, when in the presence of pus in small amounts the liquid 
becomes mucilaginous and ropy, while a gelatinous mass is obtained 
if it is abundant. 

From a clinical point of view it is most important to establish the 
source of the pus in every case of pyuria. This may at times be 
difficult, but the following data will be found of value in a differen- 
tial diagnosis : 

1 . In diseases affecting the renal parenchyma the amount of pus, 
as a rule, is small, except where a large abscess located in the kidney 
structure proper has suddenly burst into the pelvis of the kidney. 

In uncomplicated cases it is a comparatively easy matter to recog- 
nize the renal origin of the pus, as other constituents, such as renal 
epithelial cells, and especially tube-casts, are usually present at the 
same time, and, as was noted in the case of renal epithelial cells, 
leucocytes are frequently found adhering to the tube-casts, and at 
times apparently compose these entirely, when they are spoken of 
as pus-casts (see Casts). In nephritis, according to Bizzozero, the 
number of pus-corpuscles stands in a direct relation to the intensity 
and acute character of the morbid process, the greatest number 
being found in cases of acute nephritis, while in the chronic forms 
their number is usually insignificant. Whenever in the course of a 
chronic nephritis large numbers of pus-corpuscles appear, they may 
be regarded as indicating either an acute exacerbation of the disease 
or a complicating inflammation of some portion of the urinary tract. 
In such cases errors may be guarded against by carefully observing 
the number and character of the epithelial cells present at the same 
time, when it will often be found that what at first sight appears as 
an acute exacerbation of a chronic process, judging from the number 
of pus-corpuscles, is in reality a secondary pyelitis, ureteritis, or 
cystitis. 

In cases of simple renal hyperemia pus-corpuscles never occur in 
notable numbers. 

2. In pyelitis the amount of pus eliminated may vary consider- 
ably, and at times even perfectly normal urine may be voided. This 
is probably owing to the fact that the ureter of the affected side, if 
the disease is unilateral, becomes obstructed temporarily, when sud- 



608 THE URINE. 

denly large quantities may appear again. The diagnosis of pyelitis 
is often difficult, and should be based not only upon the condition of 
the urine, but upon the clinical symptoms as well. Very significant 
is the fact that the urine in pyelitis is usually acid, a point to be 
remembered in the differential diagnosis between this condition and 
cystitis, with which pyelitis is frequently confounded. A careful 
examination of the epithelial elements may also be of value, and 
should never be neglected. Bacteria in large numbers are generally 
present. 

.When pyelitis is associated with nephritis it may at times be 
almost impossible to determine the origin of the pus ; but if the rule 
set forth above is remembered, that in chronic nephritis the number 
of leucocytes is always small, it is not likely that a pyelitis will be 
overlooked, particularly if the clinical symptoms are taken into 
consideration. 

Matters may become still more complicated when a cystitis is 
accompanied by a pyelitis or a pyelonephritis. Catheterization of 
the ureters, which was first practised in the United States by the 
late Dr. James Brown, should then be resorted to, and it is highly 
desirable that this most valuable method of diagnosis should become 
common property as soon as possible. Fischl regards the presence 
of cylindrical masses composed of pus-corpuscles, formed in all 
probability in the papillary ducts, as highly characteristic of pyelitis. 

3. A pyuria referable to ureteritis can hardly be diagnosed from 
the appearance of the urine, and in suspected cases catheterization of 
the ureters should be resorted to, which may possibly elicit informa- 
tion of value. 

4. In mild cases of cystitis pus may be altogether absent, while 
in the more severe forms its presence is constant. In cystitis the 
largest amounts, referable to disease of the urinary organs, are 
observed, and are exceeded only in those rare conditions in which 
a neighboring abscess has suddenly opened into the urinary pas- 
sages. 

As the urine in cystitis is usually alkaline, and always so in the 
more severe forms, the alkalinity being due to ammoniacal fermen- 
tation, it may happen, owing to the disintegrating action of the 
ammonium carbonate upon the pus-corpuscles, that these may not 
even be demonstrable with the microscope, and that a gelatinous, 
mucoid sediment appears instead, which escapes from the vessel en 
masse when the urine is poured out. Vitali's test for pus (referred 
to on page 606) should be employed in such cases. 

5. In urethritis pus may be present in the urine in considerable 
amounts. The source of the pus is recognized by the fact that a 
drop may be manually expressed from the urethra, particularly in 
the morning upon awaking. Mucoid gonorrhoeal threads, — the 
" Tripperfaden " of the Germans, — which are largely composed of 



MICROSCOPICAL EXAMINATION OF THE URINE. 609 

pus-corpuscles, will almost always be detected iu the urine in such 
cases (Fig. 140). In order to distinguish between a simple urethritis 
and a urethritis complicated with cystitis, the urine should be 
obtained in two portions and allowed to settle. In simple urethritis 
affecting the anterior portion of the urethra the first specimen is 
cloudy, while the second one is clear. If the urethritis, however, 
has extended to the neck of the bladder, in the absence of cystitis, 
the first portion will, of course, be cloudy, while the second may 
present a variable appearance, being clear at times and cloudy at 
others. This phenomenon is explained by the fact that a portion of 
the pus contained in the posterior portion of the urethra has found 
its way into the bladder. A cystitis may, however, be excluded by 
the acid reaction of the second specimen, and the fact that the latter 
is never so cloudy as the first. In cases of urethritis complicated 
with a purulent cystitis the second portion of the urine contains at 
least as much pus as the first, and usually more, owing to the fact 
that the pus (which is heavier than the urine) falls to the floor of the 
bladder, in which case also the last drops passed will often be found 
to be pure pus. The reaction of the urine, moreover, will then 
be generally alkaline. 

6. A sudden elimination of large quantities of pus with a urine 
which up to that time has presented a normal or nearly normal ap- 
pearance may almost always be referred to rupture of an abscess into 
the urinary passages. Exceptions to this rule have been noted in 
rare instances in which large amounts of pus suddenly appeared, the 
origin of which could not be demonstrated upon post-mortem inves- 
tigation. Whether such a phenomenon, as v. Jaksch suggests, is 
dependent upon " unusual conditions favoring diapedesis " remains 
an open question. 

Enumeration of the Pus-corpuscles in the Urine. — In order to 
determine the relation existing between the degree of pyuria and 
albuminuria, as well as to watch the progress of an individual case, 
an enumeration of the number of pus-corpuscles is at times neces- 
sary. To this end, a specimen of the urine is thoroughly shaken 
and the number of corpuscles contained in one cubic millimeter 
ascertained with the aid of the Thoma-Zeiss blood-counter. Dilu- 
tion with a 3 per cent, solution of common salt is necessary when a 
preliminary examination has shown the presence of more than 40,000 
corpuscles per cbmm. A dilution of five times is usually sufficient. 
In every case one hundred squares at least should be counted. 

Some of the results which have thus been obtained are extremelv 
interesting. In cases of mild cystitis 5000 pus-corpuscles are found 
on an average in the cubic millimeter ; in cases of moderate severity 
from 10,000 to 20,000 ; while in severe cases 50,000 and even more 
may be seen. In one case of cystitis complicating carcinoma of the 
bladder Hottinger obtained 152,000 per cbmm. In the presence of 

39 



610 THE URINE. 

less than 50,000 a mere trace of albumin is found, and with 80,000-= 
100,000 only 1 pro mille is referable to this source. 1 

Red Blood-corpuscles. — The presence of red blood-corpuscles in 
the urine, constituting the condition usually spoken of as hcematuria, 
is observed only in pathological conditions, and is, in contradistinc- 
tion to hemoglobinuria (which see), a very common occurrence. 

Urine containing blood-corpuscles in notable numbers presents a 
color which may vary from a bright red to a dark brown verging 
upon black. Upon standing, a sediment of a corresponding color 
is obtained in which distinct coagula of variable size are at times 
seen. 

If the urine should contain only a small number of red corpuscles, 
however, no deviation from its normal appearance will be noted, and 
the diagnosis of hematuria can then only be made with the micro- 
scope, which should be employed in every case. The appearance of 
the red corpuscles varies greatly, being influenced especially by the 
length of time during which they have remained in the urine. In 
cases of hematuria of urethral or vesical origin it will be found 
that they have mostly retained their normal appearance fairly well, 
or have become crenated, when they may be recognized without diffi- 
culty. Other corpuscles, however, will probably also be seen which 
are no longer biconcave, but which have become spherical or shrunken 
and present an irregular outline. In cases, on the other hand, in 
which the corpuscles have remained in the urine for a longer time, 
as in hematuria of renal origin, the inexperienced will frequently be 
puzzled by the presence of bodies of the size of red corpuscles, or 
somewhat smaller, which are entirely devoid of coloring-matter, and 
appear as faint, transparent rings, often presenting a double contour, 
and in which no nucleus can be discovered. These formations are 
red blood -corpuscles from which the haemoglobin has been dissolved. 
They are usually spoken of as blood-shadows. Chemical tests are 
rarely necessary, but may be employed if doubt should arise (see 
page 507). 

Clinically it is, of course, all-important to determine the source of 
the blood. This may at times be accomplished without much diffi- 
culty by a urinary examination, but at other times it may almost be 
impossible, when the clinical symptoms and physical signs must be 
taken into consideration. 

1. Hematuria of urethral origin, due to urethritis or traumatism 
incident to catheterization, for example, is a common event, and 
readily diagnosed, as in such cases blood either escapes of itself 
from the urethra or it may be squeezed out manually. The last 
portion of the urine voided, moreover, will always be found free 
from blood, unless it is referable to disease of the neck of the bladder, 

1 E. Wunderlich, Ueber d. Werth d. Zahlung d. weissen Bl utkorperchen im Harn, 
etc., Diss., Wiirzburg, 1885, 



MICROSCOPICAL EXAMINATION OF THE URINE. 611 

when the blood appears only toward the end of micturition, or at 
least more markedly then than in the beginning. 

2. The diagnosis of vesical hsematuria is not always easily made. 
It should be remembered, however, that the blood-corpuscles here 
present a normal appearance, as has been mentioned, unless animo- 
niacal decomposition is occurring in the bladder, in which case blood- 
shadows are seen in large numbers. The blood, moreover, is less 
intimately mixed with the urine than in cases of renal haeruaturia, 
so that the corpuscles rapidly settle after the urine has been passed. 
Blood-clots of an irregular form and considerable dimensions can 
only be of vesical origin. A careful examination for the presence 
of any other morphological constituents which may be observed in 
urinary sediments, when considered in conjunction with the clinical 
symptoms, will usually lead to a correct diagnosis so far as the seat 
of the hemorrhage is concerned. Hsematuria of vesical origin may 
be due to numerous causes, among which may be mentioned diph- 
theritic cystitis, ulcers of the bladder caused by calculi and carci- 
noma, traumatism, the presence of parasites, and, more rarely, rupture 
of varicose veins in the bladder. In determining the cause of the 
hemorrhage in a given case more reliance should be placed upon the 
clinical history than upon the urinary examination. 

3. In hematuria of ureteral origin characteristic blood-coagula, 
corresponding in diameter and form to the ureters, are occasionally 
seen. Their presence, however, does not necessarily indicate that 
the blood has come from the ureters ; more frequently the hemor- 
rhage will be found to be due to disease of the pelvis of the kidney. 

4. The diagnosis of hemorrhage into the pelvis of the kidney 
must be based upon the clinical symptoms taken in conjunction with 
the results of a urinary examination. In nephrolithiasis only a small 
number of red corpuscles is usually found, which is important from 
the standpoint of differential diagnosis. 

5. Hematuria of purely renal origin is of common occurrence, and 
may be due to numerous causes. In simple hypersemic conditions of 
the organs and in acute nephritis the passage of smoky -looking urine 
containing blood-corpuscles, usually in large numbers, is thus a fairly 
constant symptom. In chronic nephritis the number of the red cor- 
puscles may be taken to indicate the intensity of the morbid process. 
Hematuria may also be due to renal abscess, nephrophthisis, renal 
carcinoma, and, in rare instances, to aneurism and embolism of the 
renal artery, thrombosis of the renal vein, etc. In the malignant 
forms of the acute infectious diseases, such as small-pox, yellow 
fever, malaria, etc., in scurvy, haemophilia, and purpura, in leukaemia, 
filariasis, and distomiasis, renal hematuria is common. It is also 
observed in cases of poisoning with turpentine, carbolic acid, can- 
tharides, and has recently also been observed in several convalescents 



612 THE URINE. 

from typhoid fever while under treatment with urotropin ; the hema- 
turia ceased with the discontinuance of the drug. 1 

6. An idiopathic form of hematuria has also been described, in 
which hemorrhage from the kidneys occurs without apparent cause. 
To this form Senator applied the term " renal haemophilia." I have 
seen three cases of this kind in which no lesion existed which could 
be made responsible for the hemorrhage. In all three the attacks of 
hematuria were invariably associated with anachlorhydria, while 
normal values were found between the attacks. Two of the patients 
were males, and undoubtedly neurasthenics. The third was a hys- 
terical chlorotic female, in whom hematemesis, pulmonary hemor- 
rhages, and melena were also at times observed. 

Hematuria of renal origin is usually recognized without much 
difficulty, as in such cases tube-casts bearing red blood-corpuscles, 
and at times apparently consisting of these altogether, as well as 
numbers of renal epithelial cells, will usually be found upon careful 
examination. The blood, moreover, is intimately mixed with the 
urine, and the individual corpuscles have mostly lost their hemoglo- 
bin and appear as mere shadows. The clinical history should, of 
course, always be taken into consideration, especially in determining 
the. primary cause of the hemorrhage. 

Urine containing red blood-corpuscles is always albuminous, so 
that it may sometimes be difficult to decide in a given case whether 
the albumin found is due solely to the presence of blood or whether 
the hematuria is complicated with an albuminuria per se. Fre- 
quently it is possible to arrive at some conclusion by comparing the 
amount of albumin with the number of the red corpuscles, the 
presence of a large amount of the former in the presence of only a 
small number of the latter indicating that the albumin is not alto- 
gether due to the blood. At other times it is impossible to gain 
information in this manner, when the only expedient left is to deter- 
mine the quantity of albumin and of iron separately, and to ascertain 
whether the amount of iron found is sufficient to combine with that 
of the albumin. As a rule, however, the presence of serum-albumin, 
aside from that contained in the blood of the urine, may be inferred 
whenever tube-casts are present, although the amount can only be 
estimated approximately in this manner. 

Tube -casts. — In various pathological conditions, and it is claimed 
even hi health, curious formations are seen in the urine, which repre- 
sent moulds of different portions of the uriniferous tubules. To these 
the term tube-casts or urinary cylinders has been applied, and it may 
be said that there is hardly a subject of greater importance in urinary 
analysis, from a clinical point of view, than that of cylindruria ; but 
it must also be admitted that notwithstanding numerous investiga- 
tions our knowledge of their nature and mode of formation is still 

1 Griffith, Milligan, and Forbes, Brit. Med. Jour., June 29, 1901. 



MICROSCOPICAL EXAMINATION OF THE URINE. 613 

defective, and the same may be said of their clinical significance. 
The term " tube-casts," however, is not altogether appropriate, as it 
is applicable to only one great division of such formations — i. e., to 
those consisting of a uniform, transparent, gelatinous matrix, to 
which other elements, such as epithelial cells, red blood-corpuscles, 
leucocytes, and salts in a crystalline or amorphous form, may acci- 
dentally have become attached — the tube-casts proper. 

From these the so-called " pseudocasts " must be sharply differ- 
entiated, a pseudocast being characterized essentially by the absence 
of a uniform matrix. Closely related apparently to the true casts are 
the so-called cylindroids — i. e., band-like formations which resemble 
the former in appearance, and like these may carry various morpho- 
logical elements as well as salts. It is thus necessary to distin- 
guish between true casts, pseudocasts, and cylindroids. Of these, 
the true casts are by far the most important. They may be 
divided into hyaline and waxy casts, the two forms being readily 
differentiated by the fact that the former readily dissolve in 
acetic acid, while the waxy casts are either not affected at all by this 
reagent, or, if so, at least not so rapidly. The latter, moreover, are 
more strongly refractive, to which property their waxy appearance 
is due ; their color is slightly yellow or yellowish gray, while the 
hyaline casts are colorless and usually very pale and transparent 1 

Mode of Examination. — Unless a urine can be examined within 
a few hours after being voided, it is well to add a small amount of 
chloroform, so as to guard against bacterial decomposition. The use 
of conical glasses is unsatisfactory, and I find it more convenient 
to keep the urine in well-stoppered bottles. Preserved with chloro- 
form it will keep almost indefinitely. Where a centrifugal machine 
is available the specimen can, of course, be examined at once. As 
soon as a sufficient amount of sediment has been obtained, a few 
drops are spread on a slide and examined, uncovered, with a low 
power. It is essential, however, to make use of the flat mirror and 
to avoid a bright light. If this is borne in mind, no difficulty what- 
ever will be found in demonstrating even the most hyaline specimens, 
though they may be present in very small numbers. In many text- 
books on urinary analysis the writers speak of the difficulty at- 
tending the search for hyaline casts, and the advice is frequently 
given to color the preparations with a drop of a dilute aqueous solu- 
tion of iodo-potassic iodide, or of some other staining reagent, such 
as gentian-violet, picrocarmin, methylene-blue, or osmic acid. This 
is unnecessary if the directions just given are strictly followed. If 
a bright light is used, however, I am willing to admit that even the 
most experienced examiner may be unsuccessful in his search. 

For the preservation of mounted specimens the following method, 

1 Eovida, see J. Moleschott, Untersuchung. z. Naturlehre d. Menschen u. d. Tkiere, 
1867, vol. xi., I. p. 182. 



614 THE URTNE. 

devised by Kronig, may be employed, though I personally prefer to 
keep the urine itself and to mount a fresh specimen when necessary. 
A drop of the sediment, best obtained by centrifugation, is spread 
on a cover-glass and allowed to dry in the air, It is then placed 
in a 10 per cent, solution of formalin, for ten minutes, rinsed in 
water, and stained for about ten minutes in a concentrated solution 
of Sudan III. in 70 per cent, alcohol. The excess of stain is 
removed by immersion for one-half to one minute in 70 per cent, 
alcohol, when the specimen is counter stained with Ehrlich's hsema- 
toxylin, rinsed in water, and mounted in glycerin. Evaporation is 
guarded against by ringing the specimen with asphaltum. The 
tube-casts are thus stained a more or less pronounced blue, the 
nuclei of the leucocytes dark blue, and any fatty granules or needles 
of fatty acids that may be present a bright red. 

I have obtained very satisfactory results by pouring a small 
amount of a 1 per cent, aqueous solution of eosin into one of the 
tubes of the urinary centrifuge, filling up with urine and then cen- 
trifugating. The supernatant fluid is poured off and the sediment 
mixed with Farrant's solution ; the specimens are finally ringed 
with asphaltum and keep for a loug time. The hyaline casts appear 
a delicate rose ; any adhering granules or cells are stained an intense 
red. Leucocytes and red cells are also well stained in this manner. 

The Farrant solution is prepared as follows : Equal parts of dis- 
tilled water, glycerin, and a saturated solution of arsenious acid 
(saturated during several weeks 7 standing) are mixed, and an amount 
of gum arabic added to occupy one-half of the bulk of the total mixt- 
ure. This is allowed to stand for several weeks (three), stirring 
daily, until all the gum has dissolved. If necessary, it is then 
filtered, which, however, is very tedious. 

Liebmann 1 recommends a mixture of 2 grammes of methylene- 
blue dissolved in 100 c.c. of a 10 per cent, solution of formalin. 
The urine is first centrifugated, the supernatant fluid is poured off, 
when a few drops of the reagent are poured on the sediment, and 
left a few minutes. The tube is filled with water, left for awhile for 
the salts to dissolve, then centrifugated again, when the formed 
elements are ready for microscopical examination. 

True Casts. — 1. Hyaline Casts (Plate XXI.). — Upon careful 
examination it will be seen that with rare exceptions the matrix of 
hyaline casts is not altogether homogeneous, as small granules may 
almost always be detected imbedded in or adhering to the matrix. As 
these granules occur in greater or less numbers, hyaline casts are 
spoken of as being finely granular (Plate XXI.), coarsely granular, 
finely dotted, etc. Should true morphological elements be detected, 
the casts are termed blood-casts, epithelial casts (Fig. 132), or pus- 
casts (Fig. 133). It would be better, however, to add the term 

1 P. Liebmann, Hospitalstidende (Copenhagen), July 30-Aug. 20, 1902. Abst. in 
Jour. Am. Med. Assoc, Sept. 20, 1902. 



PLATE XXI 



.^^& 





» 




LS. 



Hyaline, Finely Granular, and Coarsely Granular Casts. 



MICROSCOPICAL EXAMINATION OF THE URINE. 615 

hyaline in everv instance, so as to distinguish them from pseudo- 
casts, which consist of these elements entirely, and lack a uniform 
matrix. It would thus be proper to speak of hyaline epithelial casts 
hyaline blood-casts, etc., and to apply the collective term— compound 
hyaline casts — to these various subvarieties. 

Fig. 132. 




Epithelial casts. 

The nature of these various forms can probably always be made 
out without much difficulty, and even in those cases in which the 
hyaline matrix is apparently concealed beneath cellular elements it 
will usually be possible, upon closer observation, to detect a fine 
boundary-line at some portion of the structure. Not infrequently 

Fig. 133. 





Pus-casts. 



also the end of the cast will be seen to be more or less distinctly 
hyaline. In others, again, a hyaline zone may be observed along 
the sides of a central organized thread, so to speak, this being fre- 
quently seen in specimens which are very broad and long. Should 
doubt arise, however, a drop of acetic acid is added to a drop of 
the sediment on the slide ; the acid dissolves the hyaline matrix, 
the organized constituents are set free, and the differential diagnosis 
between a pseudocast and a compound hyaline cast is thus readily 
established. 

The length of hyaline casts varies greatly. It may scarcely 
exceed the breadth, on the one hand, while on the other, although 



616 



THE URINE. 



rarely, the cast may traverse the entire microscopical field. In 
breadth they vary between 0.01 and 0.05 mm. As a rule, the 
breadth of a cast is uniform throughout its entire length, but speci- 
mens are not infrequently observed in which one end tapers con- 
siderably and presents a spirally twisted appearance. This may be 
so marked that the entire cast appears transversely striated. It 
is generally supposed that this results from the adhesion of one end 
of the cast to the walls of a tubule, the lumen of which it does not 
fill, so that the free end becomes twisted in the downward course. 
A dichotomous branching of one end is also at times seen in very 
broad hyaline specimens. 

" Fatty globules are found upon the surface of granular casts 
(Fig. 134), but they also form by themselves short, strongly refrac- 
tive casts, which are often beset all over with needles of fatty crys- 
tals. These, however, are not composed exclusively of fat, but 
probably to some extent of lime and magnesium salts of the higher 

Fig. 134. 




a, Fatty casts, b and c, Blood-casts, d, Free fatty molecules. (Roberts.) 

fatty acids and allied compounds, for they are not all soluble in 
ether. They have their origin doubtless in fatty degeneration of 
the renal epithelium " (v. Jaksch). 

Granules of melanin, indigo, and altered blood-pigment may at 
times be observed in casts. Eiedel regards the occurrence of dark- 
brown casts as pathognomonic of fractures. 

2. The waxy casts (Fig. 135) may be divided iuto two groups — 
true waxy casts and amyloid casts ; but as the latter are not neces- 
sarily indicative of the existence of amyloid degeneration of the kid- 
neys, such a classification is at the present time at least of only 
theoretical interest. They are readily distinguished from the hyaline 



MICROSCOPICAL EXAMINATION OF THE URINE. 



617 



casts by the characteristics mentioned above — i. e. 9 their higher 
degree of refraction, their yellow or yellowish-gray color, and the fact 
that they are either not attacked at all by acetic acid or only very 
gradually. As a rule, only small fragments are found, but these are 
broader and more compact than the largest hyaline casts. Waxy casts 
may also contain cellular elements, crystals, and amorphous mineral 
matter; but, as a rule, such compound casts are not so commonly 
observed as are those of the hyaline variety. From the latter they 
differ furthermore in frequently presenting a cloudy appearance, 
which in some cases is undoubtedly due to the presence of innumer- 
able bacteria, and it has been suggested 
that these may be directly concerned in Fig. 135. 

their production. 

As has just been stated, some waxy 
casts give the amyloid reaction — i. e., they 
assume a mahogany color when treated with 
a dilute solution of iodo-potassic iodide, 
which changes to a dirty violet upon the ad- 
dition of dilute sulphuric acid. It should be 
remembered, however, that this reaction in 
casts does not necessarily indicate the exist- 
ence of amyloid disease of the kidneys, as 
the reaction may be absent on the one hand 
in this condition, and present on the other 
where amyloid degeneration does not ex- 
ist. This curious phenomenon is usually 
explained by assuming that such casts 
have remained in the uriniferous tubules 
for a long time, and have there undergone 
certain chemical changes analogous to the 
so-called " amyloid metamorphosis " of 
old precipitates of fibrin, and it is indeed 
possible that waxy casts are originally 
hyaline. Frerichs has pointed out that 
fibrin which has remained in the uriniferous 
ctnbules for a long time becomes denser and 
yellowish in appearance, which would explain the fact that these 
casts are only with difficulty attacked by acetic acid. 1 

Before leaving this subject it should be stated that " cast-like " 
formations consisting entirely of amorphous urates are not infre- 
quently encountered in urines, and according to Leube they may be 
obtained from any urine if it is concentrated in a vacuum at a tem- 
perature of 37° to 39° C. 2 Students frequently regard such forma- 
tions as coarsely granular casts, an error which may be guarded against 
if the characteristics of hyaline casts set forth above are borne in mind. 

1 Eovida, loc. cit. Kobler, Wien. klin. Wock., 1890, vol. iii. pp. 531, 557, 574, 576. 

2 Leube, Zeit. f. klin. Med., 1887, vol. xiii. 





Different forms of waxy 
casts: a, With a coating of" 
urates, b, Waxy cast covered 
with crystals of calcium oxal- 
ate, c, Fragments of waxy 
casts, (v. Jaksch.) 



618 THE URINE. 

Bacteria (in cases of infectious pyelonephritis), haematoidin, and 
granular detritus frequently occur grouped in a cast-like manner ; 
their nature is readily ascertained, as in the case of the so-called 
urate casts just described. 1 

Pseudocasts, consisting of epithelial cells or blood-corpuscles and 
fibrin, are not often found in urinary sediments. The epithelial 
pseudocasts are probably seen only in cases of desquamative nephritis, 
and, unlike true casts, are hollow, the epithelium of the uriniferous 
tubules being thrown off en masse. Blood-casts (Fig. 134) consist 
of fibrin, within the meshes of which red corpuscles are generally 
found ; these either present a normal appearance or occur as mere 
shadows, owing to the fact that their haemoglobin has been dissolved. 
They are seen whenever extensive hemorrhage has taken place in the 
renal parenchyma, and are far more frequently observed than the 
epithelial pseudocasts. Hyaline casts are probably always met with 
in urinary sediments in which pseudocasts are found, and may be 
readily distinguished from the latter even when beset with numerous 
epithelial cells or red corpuscles (see above). 

Cylindroids (Fig. 136) resemble hyaline tube-casts somewhat in 
general appearance, but differ from them in being much larger and 
band-like. Like true casts, they have a uniform breadth, and are 
often beset with crystals and cellular elements, such as leucocytes, 
red corpuscles, and epithelial cells. They are readily dissolved by 
acetic acid, thus differing from the mucous cylinders or pseudo- 
cylinders (Fig. 137) which may be observed in any urine containing 
mucus ; the latter probably never contain morphological or mineral 
constituents, and are never of uniform breadth throughout their 
length. The cylindroids proper are undoubtedly of renal origin 
and closely related to true casts ; formations are indeed not infre- 
quently seen in which a tube-cast terminates in a cylindroid at one 
or both ends. 2 

Formation of Tube-casts. — Several hypotheses have been advanced 
to explain the formation of tube-casts — reference is here had only to 
true casts, and not to pseudocasts, the origin of which is sufficiently 
obvious — and until recently it was quite generally accepted that they 
consist of coagulated albumin which has transuded into the tubules. 
According to this view, a cylindruria would always be indicative of 
the existence of albuminuria. In Neubauer and Vogel's Urinary 
Analysis (ninth edition) it is stated that " as to the significance 
of tube-casts, it must be remembered that these, according to our 
present knowledge, consist of albumin, which coagulates under the 
influence of the acid reaction of the urine, in the renal paren- 
chyma, in a peculiar hyaline manner. They represent merely a 

1 Martini, Arch. f. klin. Chir., 1884, vol. xvi. p. 157. v. Jaksck, Deutsch. med. 
Woch., 1888, vol. xiii. Nos. 40 and 41. 

2 Bizzozero, loc. cit. Thomas, Arch. f. Heilk., 1870, vol. xi. p. 130. Pollaku. 
Torok, Arch. f. exper. Path. u. Pharmakol.. 1888, vol. xxv. p. 87. 



MICROSCOPICAL EXAMINATION OF THE URINE. 



619 



solidified portion of the albumin held in solution by the urine ; their 
elimination essentially indicates the existence of an albuminuria." 
More recently, however, it has been suggested that tube-casts are 
the product of a faulty metamorphosis, or of inflammatory irrita- 
tion of the renal epithelium, and that a secretion from these cells or 



Fig. 136. 




Fig. 137. 



a and b, Cylindroids from the urine in 
congested kidney, (v. Jaksch.) 



Mucous cylinders. 



a disintegration of their protoplasm occurs, which results in the 
formation of cylindroids or true casts. 1 

Clinical Significance of Tube-casts. — Formerly the occurrence of 
tube-casts in urine was held to indicate the existence of nephritis. 

1 See also Eindfleisch, Lehrbuch d. path. Gewebelehre, Leipzig, 3875, p. 438. 
Langhans, Virchow's Archiv, 1879, vol. lxxvi. p. 85. Kovida, loc. cit. Kobler, loc. 
cit. Ribbert, Centralbl. f. d. med. Wiss., 1880, vol. xix. p. 305. 



620 THE URINE. 

This view has been abandoned, however, for the same reasons which 
led to the rejection of the theory that albuminuria invariably indi- 
cates Blight's disease (see above). 

The statement is frequently made in text-books that tube-casts 
may occur in the urine of perfectly healthy individuals, following 
severe muscular exercise, cold baths, etc. — in short, all stimuli which 
may cause the appearance of albumin in apparently normal individ- 
uals. It has been indicated elsewhere (see Functional Albuminuria), 
however, that such stimuli cannot be regarded as " physiological " 
in every instance, and the presence of tube-casts in the urine similarly 
should be regarded as a pathological event. 1 

It is not necessary in this connection to enumerate the various 
diseases in which cylindruria is observed, as they are the same as 
those which give rise to albuminuria ; and just as a nephr angiogenic 
albuminuria is more frequently observed than a nephritidogenic albu- 
minuria, so also is the presence of tube-casts in the urine more fre- 
quently due to circulatory disturbances in the kidneys than to true 
nephritis. In every case in which tube-casts occur in the urine it 
may be assumed that the accompanying albuminuria is, to a certain 
extent at least, of renal origin. 

While the existence of cylindruria is not necessarily associated 
with definite pathological alterations of the renal parenchyma, this 
statement should be restricted to the occurrence of purely hyaline 
casts, and their presence in only small numbers. A few renal epi- 
thelial cells may be found at the same time, occurring either free in 
the urine or adhering to the casts, but never presenting an atrophic 
or otherwise altered appearance in the absence of definite renal lesions. 
The presence of compound hyaline and coarsely granular casts, as 
well as of waxy and amyloid casts, on the other hand, may probably 
always be regarded as indicating definite changes in structure, so that, 
so far as the diagnosis of nephritis is concerned, a microscopical 
examination of the urine will furnish information of more value 
than the simple demonstration of albumin. 

Hyaline casts are those most frequently seen, — reference is here 
had only to the purely hyaline or, at least, but .faintly granular 
form, — and are found in all conditions in which albuminuria occurs. 
When present in only small numbers, and particularly when occur- 
ring but temporarily in the urine, it may be assumed, in the absence 
of other symptoms pointing to renal disease, that we are dealing 
with a mild circulatory disturbance of the kidneys. Renal epithelial 
cells are absent, or present, in only small numbers. The albumin- 
uria at the same time is trifling. If, however, hyaline casts are 
continuously present in large numbers, and if the amount of albumin 
exceeds a trace, the existence of a nephritis may usually be inferred. 

1 Nothnagel, Deutsch. Arch. f. klin. Med., 1874, vol. xii. p. 326. Burkhart, Die 
Harneyliiider, 1884. Fischel, Prag. Vierteljahrschr., 1878, vol. cxxxix. p. 27. 



MICROSCOPICAL EXAMINATION OF THE URINE. 621 

In such cases granular casts and compound hyaline casts, particu- 
larly the former, will be found if the nephritis is chronic, while in 
the acute form the hyaline type prevails. Should blood-casts be 
present at the same time, the probabilities are that we are dealing 
with an acute nephritis or an acute exacerbation of. a chronic process ; 
in the latter case coarsely granular casts will also be present in large 
numbers. 

Waxy casts always indicate a chronic or, at least, a subacute 
process. The fatty casts described by Knoll and v. Jaksch " are 
most commonly associated with subacute or chronic inflammations 
of the kidney of protracted course, with a tendency to fatty degen- 
eration of the renal tissue. Post-mortem examination has shown 
that they form most frequently in cases of large white kidney. In 
some cases in which they were present, however, the organ was 
found to be more or less contracted ; but when this was so, it was 
invariably far advanced in fatty degeneration." 

It has been stated that from a careful examination of the renal 
epithelial cells it is often possible to determine whether an inflamma- 
tory process affecting the kidneys is at the same time complicated 
with degenerative changes. As a matter of fact, the cells found on 
the tube-casts under such conditions no longer present a normal 
appearance, but are shrunken and atrophied, and in cases of fatty 
degeneration studded with fatty granules. Epithelial casts, in the 
absence of distinct changes affecting the renal parenchyma, are prob- 
ably never seen. 

The occurrence of pus-casts presupposes the existence of suppura- 
tive inflammation in the kidneys, while the presence of only a small 
number of leucocytes on hyaline casts may be observed in the ordi- 
nary forms of nephritis, and particularly in the acute form. 

The pathological significance of the so-called amyloid casts and 
pseudocasts has already been considered. 

Cylindroids are present whenever hyaline casts are seen, and have 
essentially the same import. They are said to occur most frequently 
in the urine of children. 

So far as the constancy with which tube-casts occur in the urine 
in nephritis is concerned, it is well known that in the chronic inter- 
stitial form of the disease they, as well as the albumin, are frequently 
absent for a long time, so that it may only be possible to make the 
diagnosis from the clinical history and the physical signs. It is a 
well-known fact, moreover, that pathological alterations of the kid- 
neys, particularly in men past middle age, are observed again and 
again in the post-mortem room, where a previous examination of the 
urine showed no evidence of the existence of renal disease. In the 
acute and subacute forms of nephritis, as well as in the ordinary 
parenchymatous form, tube-casts are probably always found, and it 
would further appear that acute circulatory disturbances affecting 



622 



THE URINE. 



the renal parenchyma quite constantly lead, not only to albuminuria, 
but also to cylindruria. 

Spermatozoa. — Spermatozoa, for a description of which the reader 
is referred to the chapter on the Semen, are frequently observed in 
the urine of healthy adults, and are quite constantly met with in the 
first urine passed after coitus or nocturnal emissions, when their 
presence is, of course, of no significance (Fig. 138). Such urines 
are always cloudy, but it is impossible to recognize the source of the 
turbidity by simple inspection. 

A sediment composed of phosphates is popularly often regarded 
as due to semen, and no doubt every physician has seen patients, 
-—usually sexual neurasthenics, — who were greatly alarmed at find- 
ing a white deposit in the chamber, and who imagined themselves 



Fig. 138. 




Human spermatozoa. 



" sufferers from loss of manhood." The microscope is necessary in 
every case to determine the presence of spermatozoa. 

In females semen may be found in the urine whenever the external 
genitals have been polluted during or after coitus, as well as in the 
exceptional cases in which connection has been effected by the urethra. 
From a medico-legal standpoint the discovery of spermatozoa in the 
urine of women may be of the greatest importance, but otherwise it 
is without significance. 

In a few instances it is stated that trichomonads have been mis- 
taken for spermatozoa. I am convinced, however, that such an 
error can only occur if the observer is totally unacquainted with the 
subject under consideration. 

In pathological conditions spermatozoa are not infrequently found 
in the urine. In cases of obstinate constipation, owing to pressure 



MICROSCOPICAL EXAMINATION OF THE URINE. 623 

of hard scybalous masses upon the seminal vesicles, a partial evacu- 
ation of semen may occur, which may or may not be accompanied 
by sexual excitement. Horowitz has pointed out that a discharge 
of semen may be noted in cases of peri-urethral abscess with per- 
foration into the ejaculatory ducts, giving rise to spermato-cystitis, 
the condition being due to a tight stricture of the urethra with 
dilatation beyond the constricted portion. I have observed a case 
of cystitis in which spermatozoa could almost always be detected 
in the urine. An operation revealed a tight stricture of the urethra 
and a sacculated bladder ; the constant passage of semen was 
apparently owing to the irritating action of the ammoniacal urine. 
It should be noted that in this case, as well as in those in which 
semen is frequently passed during the act of defecation in the absence 
of sexual excitement, no deleterious effects referable to such loss were 
noted. In the urine voided during and after epileptic and, more 
rarely, hystero-epileptic seizures spermatozoa may be found. Such 
an event is undoubtedly due to muscular spasm, and is identical in 
origin with the emission of semen observed so frequently in the 
death agony, and especially during strangulation. 

In certain spinal diseases semen may be found in the urine, and 
Furbringer relates a case in which, following fracture and dislocation 
of the vertebral column, with partial destruction of the middle dorsal 
cord, spermatorrhoea associated with partial erection occurred thirty 
hours later, and continued until death, which took place after three 
days. 

More important is the loss of semen noted in cases of true sperma- 
torrhoea due to venereal excesses or masturbation, when spermatozoa 
may be found almost constantly, and the diagnosis indeed will often 
be dependent upon such an observation. 

So far as the question of sterility in the male is concerned, reliance 
should not be placed upon an examination of the urine, but the semen 
should be obtained as soon as possible after ejaculation, and exam- 
ined as indicated elsewhere. 

Parasites. — Vegetable Parasites. — It has been shown by numerous 
investigations that bacteria are always present both in the male and 
female urethra, and that they may at times gain entrance to the 
bladder. The weight of evidence, however, is in favor of the view 
that the urine intra vesicam is under normal conditions free from 
micro-organisms, and that any bacteria which may have found their 
way into the bladder are rapidly killed in healthy individuals. In 
every urine, on the other hand, that has been exposed to the air, 
bacteria are always present. Whenever, then, it is desired to deter- 
mine whether or not the urine of the bladder contains micro-organ- 
isms, every precaution should be taken to guard against accidental 
contamination. To this end, the following method should be em- 
ployed : if the patient is a male, he is instructed to hold his urine 



624 THE URINE. 

until a fairly large amount has accumulated. The glans is then 
thoroughly washed with soap and water, and further cleansed with 
cotton soaked in mercuric chloride solution (1 : 1000). The fossa 
navicularis is also thoroughly cleansed with the same solution. The 
urine is then voided under as great pressure as possible. The first 
portion (about 100 c.c.) is thrown away, and the second received in 
a sterilized vessel, when cultures should be made at once, agar or 
gelatin plates being inoculated with 1 or 2 c.c. of the urine. In 
the female the vulva is cleansed with soap and water, and the urethral 
aperture disinfected with bichloride solution. After then washing 
with sterilized water and drying with sterilized cotton the urine is 
evacuated through a sterilized metallic or glass catheter, and received 
in a sterilized vessel. Brown describes the method which is in use 
in Dr. Kelly's department at the Johns Hopkins Hospital as follows : 
the external urethral orifice being carefully cleansed with mercuric 
chloride solution, followed by sterile water, a sterilized glass catheter, 
whose external end is covered by a sterile rubber cuff, extending 
several centimeters beyond the end of the catheter, is introduced, 
the fingers of the operator being allowed to touch only the distal end 
of the rubber cuff. The urine is allowed to flow for a short time, 
when the rubber cuff is pulled off by traction on its distal end. 
A small amount of urine is then collected in a sterile test-tube, 
and the cotton plug immediately inserted. BroAvn states that an 
extended series of experiments with normal urines 
Fig. 139. h as shown that this method is absolutely reliable. 1 



CK x - O Of the bacteria which may be found in every 

niacal fermentation is largely due to its presence 



\**v/ — j urine that has been exposed to the air, the Miero- 
£s^ .^'V? coccus urece is of special interest, as ammo- 

V LA./ —J"* • 1 r» ,,• • 1 IT , • , 



-*"^ When fermentation has commenced, it is readily 

Micrococcus urete. recognized, occurring in almost pure culture upon 
the surface of the urine, mostly in the form of 
characteristic chains (Fig. 139). The individual coccus is colorless 
and quite large, so that it may be mistaken by beginners for a blood- 
shadow. 

It is a common error to infer from the occurrence of ammoniacal 
decomposition very soon after micturition that this process has already 
begun in the bladder. It should be remembered that urine may un- 
dergo fermentation, particularly in warm weather, shortly after having 
been voided, and especially if the vessel employed is not perfectly 
clean and the urine has been exposed to the air. The diagnosis of 
ammoniacal fermentation in the bladder should hence only be made 
when the presence of ammonia can be demonstrated in the urine 
immediately upon being voided. 

Under pathological conditions various pathogenic bacteria may be 

1 T. R. Brown, loc, dt 



MICROSCOPICAL EXAMINATION OF THE URINE. 625 

found in the urine. Their presence usually indicates the existence 
of definite changes in the renal parenchyma, although these changes 
are not necessarily of an inflammatory character. Pyogenic cocci 
are especially prone to settle in the kidneys, and there give rise to 
focal inflammations ; but even in the absence of such lesions they are 
frequently found in the urine. In all forms of infectious nephritis 
an abundant elimination of bacteria may generally be observed, v. 
Jaksch states that in erysipelas the bacteriuria and nephritis dis- 
appear, together with the cessation of the disease, and in various 
suppurative processes taking place in the body the specific bacteria 
disappear from the urine within twenty-four to forty-eight hours 
after evacuation of the pus. 

Most interesting observations on the occurrence of bacteria in the 
urine of nephritic patients have been reported by Engel. Thirty- 
one cases were examined. In sixteen the Staphylococcus albus and 
aureus were found, in eight pyogenic streptococci, in four the tubercle 
bacillus, in five the Bacillus coli communis, and in one the typhoid 
bacillus, while negative results were obtained in only two' instances. 
In the same series Engel also found a pyogenic coccus in seventeen 
cases. This coccus was larger than the known forms ; it could be 
stained according to Gram's method, and did not liquefy gelatin. 
Intravenous injections of large numbers of the organism caused 
nephritis in rabbits. 

In pneumonia and pneumococcus infections in general the corre- 
sponding diplococcus may be found, and in erysipelas and strepto- 
coccus infections streptococci. Very common is the presence of 
the Bacillus coli communis in cases of pyelonephritis ; it is usually 
found in pure culture, but is at times associated with the Staphy- 
lococcus aureus and the Proteus vulgaris. In some instances the 
latter organism has also been met with in pure culture. In scar- 
latina streptococci have been found in a large percentage of cases ; 
the urine was then more often albuminous than non-albuminous. 

Of great interest is the frequent occurrence of the typhoid bacillus 
in the urine of typhoid fever patients. Bouchard 1 in 1881 drew 
attention to the elimination of the bacillus through this channel, and 
stated that he was able to demonstrate its presence in 50 per cent, 
of his typhoid fever cases. Other observers were less successful, but 
with improving technique and more general investigation a larger 
number of positive results is being obtained every year. 2 At the 
present time it may be said that the typhoid bacillus can be found in 
the urine of from 20 to 30 per cent, of all typhoid fever patients. 
The organism usually appears in the second or third week of the 
disease, and may persist for months and even years. When present 

Bouchard, Rev. de Med., 1881, p. 671. 

2 For an account of the literature, see T. R. Brown, "Cystitis due to the Typhoid 
Bacillus," etc., Med. Record, March 10, 1900. 

40 



626 THE URINE. 

it usually occurs in pure culture, and often the bacilli are so numerous 
as to render cloudy a freshly voided specimen of urine. Symptoms 
of cystitis and marked renal involvement often occur, but in a consid- 
erable number of cases there are no indications of local disease. The 
elimination of the organism in the urine is of no prognostic significance, 
but is important from the standpoint of prophylaxis. Of special 
interest is the fact that the organism may at times be found in the 
urine although the patient is not the subject of typhoid fever at the 
time. Brown 1 thus reports the case of a woman in whom a cystitis 
developed on the ninth day following an abdominal operation, and 
in whom it was thought that the typhoid bacillus was accidentally 
introduced by the catheter. The patient had had typhoid fever 
thirty-five years previously. Young 2 gives the history of a patient 
in whom cystitis developed during an attack of typhoid fever, owing 
to infection with the typhoid bacillus. The organism could still be 
demonstrated in the urine after seven years. A double infection 
with the gonococcus subsequently occurred, and four months later 
typhoid bacilli and gonococci were both present in considerable 
numbers. Cystoscopic examination showed a chronic ulcerative 
cystitis. Two additional cases of chronic cystitis due to the typhoid 
bacillus are reported. 

The bacillus may be isolated and identified according to the usual 
methods (see page 325). 

In cases of paratyphoid fever the corresponding bacilli may be 
found in the urine. 

Very important further is the fact that in tubercular disease of the 
urinary organs tubercle bacilli may be found in the urine. The 
search for them, however, is always tedious and frequently fruitless. 
In suspected cases it is best to centrifugate the urine, and to spread 
the sediment upon slides or cover-glasses. The preparations are then 
fixed by heat, and are best stained with Pappenheim's reagent (see 
page 357). Grethe's method, which was formerly used to differentiate 
the two, is less reliable. With this method the specimens are stained 
with a concentrated alcoholic solution of fuchsin, the staining fluid 
being brought to the boiling-point on the slide. T ne y are then washed 
in water and counterstained with a concentrated alcoholic solution of 
methylene-blue without the application of heat. The excess of stain 
is washed off, when the preparations are dried with filter-paper and 
examined as usual. As with Pappenheim's method, the tubercle 
bacilli are colored red, while the other morphological elements which 
may be present, including the smegma bacillus, are stained blue. 
The usual methods of staining are not admissible, as the smegma 
bacillus, which may also be present in the urine, is likewise stained, 
and may readily be mistaken for the tubercle bacillus. 

1 Loc. cit. 

2 H. H. Young, " Chronic Cystitis due to the Bacillus Typhosus," Maryland Med. 
Jour., Nov., 1901, p. 456. 



PLATE XXII. 



s * 









" m 



• t *i 



L. SCHMIDT, FEC. 



Urethral Discharge from a Case of Gonorrhoea, showing Gonoeoeei Enclosed, in 

Pus Corpuscles, and Lying Free in the Discharge. Stained with 

Methylene Blue. (Personal Observation.) 



MICROSCOPICAL EXAMINATION OF THE URINE. 627 

If, in suspected cases, notwithstanding repeated examination and 
the preparation of numerous specimens, tubercle bacilli are not found, 
it is best to inject a few drops of the sediment into the anterior 
chamber of the eye of a rabbit, and to watch for the development 
of miliary tubercles in the iris. 

The number of bacilli which may be found in the urine in tuber- 
cular disease of the urinary organs is extremely variable. Fre- 
quently none at all are found, notwithstanding careful search ; in 
other cases they are present in small numbers ; while in still others 
they are extremely numerous, and are often bunched to form par- 
ticles visible to the naked eye. 

Isolated tubercle bacilli have also been found in the urine in cases 
of acute miliary tuberculosis, in the absence of renal changes ; such 
observations, however, are rare. Fouler ton and Hillier 1 claim that 
the tubercle bacillus, virulent to guinea-pigs, occurs in the urine in 
50 per cent, of far advanced cases of pulmonary tuberculosis, and as 
a rule in the absence of any renal lesion. Their observation thus 
accords with the findings of others that the organism in question 
infests the blood more commonly than was formerly supposed. As 
a rule, however, but few organisms were' obtained. 

Gonococci may be found in urinary sediments enclosed in pus 
cells, and can be demonstrated by preparing smears and staining 
with a basic dye or with the eosinate of methylene-blue solution. 
In the so-called gonorrhoea! threads they can often be found years 
after the infection (Plate XXII. ). 

In cases of bubonic plague Kitasato's coccobacillus may be found 
in the urine. 

In cases of cystitis a great variety of micro-organisms has been 
met with in the urine. Among the more important may be men- 
tioned the staphylococcus aureus, albus, and citreus, streptococci, 
the bacillus coli communis, the bacillus pyocyaneus, the bacillus 
typhosus, the proteus vulgaris, the gonococcus, etc. In many cases 
of cystitis organisms are found, moreover, which are apparently non- 
pathogenic, and are capable of causing the formation of hydrogen sul- 
phide from certain sulphur bodies of the urine (see Hydrothionuria). 

Actinomyces kernels may be observed in the urine when the 
disease in question has attacked the genito-urinary tract or when 
the organism has found its way into the urine from other organs. 

In conclusion, reference should be made to the occasional occur- 
rence of a form of bacteriuria which is not associated with any 
pathological process, and has hence been termed idiojmthic bacteriuria. 
Of its causation and significance nothing is known, but it is pos- 
sible that in these cases a few bacteria enter the bladder either 
through the anterior rectal wall or are eliminated through the kid- 
neys from the blood-current. Finding a suitable medium for their 

1 A. G. Foulerton and W. T. Hillier, Brit. Med. Jour., Sept. 21, 1901. 



628 THE URINE. 

growth in the urine, they here multiply and may thus be constantly 
present. Of late, the Bacillus lactis aerogenes has been found in 
such a case. The diagnosis " idiopathic bacteriuria " should, of 
course, only be made if every possible source of contamination of 
the urine can be definitely excluded. 1 

Urines containing bacteria in large numbers are always cloudy, 
and usually present an acid inaction when voided unless cystitis 
exists at the same time. Attention is directed to their presence by 
the fact that such specimens cannot be cleared by simple filtration. 

Yeast-cells in large numbers are usually only seen in urines con- 
taining sugar. Whenever a chemical examination has not been made 
their demonstration will be of importance, as suggesting the pos- 
sible existence of glucosuria. 

Moulds are usually seen in old diabetic urines after alcoholic fer- 
mentation has taken place, but they may also occur, though far less 
frequently, upon the surface of putrid urines that have contained 
no sugar. 

The uriuary sarcina which is at times met with is smaller than 
the sarcina of the gastric contents, but closely resembles it in appear- 
ance. It is of no clinical significance. 

Whenever a urine is to be examined bacteriologically, special pre- 
caution should be taken to guard against its accidental contamination. 
The safest procedure, of course, is to obtain the urine by suprapubic 
puncture. This is, however, only exceptionally necessary, and as a 
general rule the method of disinfection which I have described 
above (see page 623) will suffice. 

Animal Parasites. — The organism which Hassal saw in a urine 
that had been " freely exposed to the air " and was alkaline, and 
which he termed Bodo urinarius, was in all probability an infusorial 
monad and of no pathological significance. Salisbury was the first 
to point out that the Trichomonas vaginalis of Donn6 may at times 
occur in the bladder, but he gave no detailed account of his cases. 
Kiinstler, Marchand, Miura, and Dock subsequently reported cases in 
which flagellate protozoa were found, and modern research leaves no 
doubt that the organisms described by these observers are identical 
with the trichomonas of Donne. In Miura's case the habitat of the 
parasite was the urethra, and an examination of the patient's wife 
revealed the presence of similar organisms in the vagina. Kiinstler' s 
case was one of pyelitis following cystotomy. Marchand' s patient 
had a fistula in the perineum following suppuration in the pelvis, of 
unknown origin ; cystitis did not exist. Dock's case was associated 
with hematuria. 2 During the past few years I have seen the same 
organism in seven cases, two of which occurred in the practice of Dr. 

1 Roberts, "On Bacilluria," Trans. Internat. Med. Cong., London, 1881, vol. ii. p- 
157. Schottelius u. Eeinhold, Centralbl. f. klin. Med., 1886, vol. viii. p. 635. Ross, 
Banmgar ten's Jahresber., 1891, vol. vi. p. 360. 

2 Dock, Am. Jour. Med. Sci., January, 1896. 



MICROSCOPICAL EXAMINATION OF THE URINE. 629 

W. M. Lewis, of Baltimore. Six were women, and I have no doubt 
that the parasite found its way into the bladder from the vagina, 
Avhere it could be demonstrated in two instances. Curiously enough 
a history of hematuria was obtained from four of the six patients. 
In two cases the urine contained blood at the time of the examina- 
tion. In one case there was evidence of nephritis ; cystitis did not 
exist. The number of the parasites was variable, and in five cases 
large. 

Balz observed numerous amoebae in the turbid urine of a girl the 
subject of phthisis, which he described as being of larger size than 
the amoeba coli. 

In cases of Bilharzia disease the ova of the parasite (see page 197) 
are probably invariably encountered in the urine together with some 
blood. Sometimes the entire bulk of the urine is blood-tinged, but 
more often only the last few drops contain blood, and in these last 
drops the eggs of the parasite will also be found. In doubtful cases 
it is always best to examine this portion. The eggs are readily seen 
with a low power (see Fig. 140). 

Fig. 140. 



mmm^. 







A gonorrhoeal thread. 

Filaria embryos may be found in the urine in cases of filarial 
chyluria. They should be looked for in the coagulum, a bit of 
which is teased out and pressed between two slides. 

Billings and Miller 1 have recently reported the possible occur- 
rence of the Anguillula aceti in the urine, in cases in which the urine 
is collected in bottles that had contained old vinegar. The worm 
closely resembles the Anguillula stercoralis. Stiles has made a 
similar observation. 

Echinococcus hooklets and fragments of cysts may also be found, 
and in rare instances ascarides find their Avay into the urinary pass- 
ages when a fistulous opening exists between the rectum and the 
bladder. Bothriocephalus linguloides (Leuckart) was found in the 
urine in a case occurring in Eastern Asia. Eustrongylus gigas is 
1 Billings and Miller, Trans. Assoc. Am. Phys., 1902, p. 161. 



630 THE VRINE. 

likewise found very rarely. Moscato records one case in which 
chyluria existed at the same time. In Clark's case, which was 
recently reported in the United States, the passage of the worm was 
accompanied by hematuria. 

Tumor-particles. — Tumor-particles are so rarely seen in the 
urine that a detailed account of their occurrence may be omitted, 
particularly as it is seldom possible to base the diagnosis of tumor 
upon the presence of fragments in the urine, the clinical history and 
the physical signs being usually sufficient to reach a satisfactory 
diagnosis. 

Foreign Bodies. — Of foreign bodies which may be found in the 
urine may be mentioned particles of fat, fibres of silk, linen, and 
wool, etc. ; in short, material the presence of which is owing to the 
use of unclean vessels for reception of the urine. Fecal matter may 
be passed by the urethra ; such an occurrence, of course, always in- 
dicates the existence of an abnormal communication between the 
bowel and the urinary passages. Hair derived from a dermoid cyst 
may similarly be found. In hysteria foreign bodies of almost any 
kind, such as hair, teeth, fish-bones, wood, etc., and even snakes and 
frogs, may be shown the physician as having been passed in the urine. 
I had occasion to examine " gravel " " passed " from time to time by 
a hysterical patient in large amounts, " every attack being accom- 
panied by agonizing pains shooting down into the lower abdomen" ; 
the gravel upon examination proved to be mortar, obtained from the 
cellar of the patient's house. 



CHAPTEE VIII. 

TRANSUDATES AND EXUDATES. 

In health the so-called serous cavities of the body contain very 
little fluid, and quantities sufficient for analytical purposes can nor- 
mally only be obtained from the pericardial sac. In pathological 
conditions, on the other hand, large accumulations of fluid may be 
observed, not only in the serous cavities, but also in the areolar con- 
nective tissue, beneath the skin, and beneath the muscles. When 
due to circulatory disturbances, a hydremic condition of the blood, 
or an insufficient elimination of water through the kidneys, such 
accumulations of fluid are spoken of as transudates, while the term 
exudates is applied to similar accumulations of inflammatory origin. 

Clinically, it is frequently difficult to distinguish between trans- 
udates and exudates, and large ovarian, pancreatic, and hydatid 
cysts, as well as cystic kidneys, may at times be mistaken for ascites. 
In such cases a careful chemical and microscopical examination of 
the fluid in question may be of decided value. Very frequently, 
moreover, it is possible only in this manner to determine the nature 
of the disease, and the free use of the trocar and the aspirating- 
needle in diagnosis cannot be too strongly advocated. 

TRANSUDATES. 

General Characteristics. 

Transudates are usually serous in character, when they present a 
light-straw color ; at times, however, owing to admixture of blood, 
they have a reddish tinge, and are then said to be sanguineous ; in 
rare instances they are chylous. 

Specific Gravity. 

The specific gravity varies somewhat according to the origin of 
the fluid, but is usually lower than that of serous exudates occurring 
in the same cavities — one of the most important points of difference 
between the two kinds of fluid. Thus, in acute pleurisy the specific 
gravity of the exudate is usually higher than 1.020 ; and in chronic 
pleurisy, if an accumulation of pus exists at the same time, higher 
than 1.018, reaching even 1.030. In transudates into the pleural 
cavity, on the other hand, referable to circulatory disturbances, for 

631 



632 



TRANSUDATES AND EXUDATES. 



example, as in cases of hepatic cirrhosis or cardiac insufficiency, the 
figures obtained are usually lower than 1.015. Transudates of peri- 
toneal origin similarly present a specific gravity varying between 
1.005 and 1.015, while that of exudates frequently reaches 1.030. 

As the chemical composition, in so far as the mineral constituents 
and extractives are concerned, is practically the same in both classes 
of fluid, the difference in the specific gravity appears to be essen- 
tially due to the amount of albumin present, viz., serum-albumin and 
serum-globulin. It may be demonstrated, as a matter of fact, that 
exudates contain far more albumin than transudates, the amount 
varying between 4 and 6 per cent, in the former, as compared with 
1 and 2.5 per cent, in the latter. The largest amounts of albumin 
in transudates are found in those of pleural origin, while in oedema 
not more than 1 per cent, is usually present. 

In the table below, taken from Reuss, the relation between the 
percentage-amount of albumin and the corresponding specific gravity 
is shown. Reuss suggests the following formula for the purpose 
of determining from the specific gravity the amount of albumin in 
transudates and exudates : 

E=\ (#— 1000) —2.8, 

in which E indicates the percentage-amount of albumin and S the 
specific gravity taken by means of an accurate urinometer. 



Specific gravity. 


Albumin. 


Specific gravity. 


Albumin 


1.008 


... 0.2 


1019 


. . . 4.3 


1.009 


... 0.6 


1.020 


... 4.7 


1.010 


. ... -1.0 


1.021 


... 5.1 


1.011 


... 1.3 


1.022 


... 5.5 


1.012 


... 1.7 


1.023 


... 5.8 


1.013 


... 2.1 


1.024 


. . . 6.2 


1.014 


. . . 2.5 

... 2.8 


1.025 


. . . 6.6 


1.015 


1.026 


... 7.0 


1.016 ...... 


... 3.2 


1.027 


... 7.3 


1.017 


... 3.6 


1.028 


... 7.7 


1.018 


... 4.0 







The following table shows the percentage-amount of albumin 
obtained by Runeberg in ascitic fluid under various pathological 
conditions : 



0.21 



Average Maximum. Minimum. 

Hydnemia (Bright' s disease, tuberculosis, 
etc., with amyloid degeneration) . . . 

Portal stasis (referable to hepatic cirrhosis 

or stenosis) .... 0.97 

General venous stasis (referable to or- 
ganic heart-disease) 1.67 

Carcinoma of the peritoneum (compli- 
cated with carcinoma of the stomach). 3.51 

Chronic peritonitis (one case complicated 

with heart-disease) 3.71 



0.41 



2.68 



2.30 
5.42 



0.03 



0.37 



0.84 



2.70 



4.25 



3.36 



A great deal of stress was formerly laid upon these factors in the 
diagnosis of transudates from exudates, but a careful study of the 



TRANSUDATES. 633 

available data goes to show that after all they are uncertain guides. 
More reliable information is gained from the chemical study of the 
albumins in accordance with the suggestion of Umber more espe- 
cially (see page 638). 

The fact that transudates do not coagulate spontaneously in the 
absence of blood may further serve to distinguish them from exu- 
dates, in which a coagulum is frequently observed after standing 
for twenty-four hours. JSot much reliance should be placed upon 
this point of difference, however, as exudates likewise do not always 
coagulate, and clotting of transudates in the presence of blood may 
take place within the body. 

Literature. — Reuss, Deutsch. Arch, f. klin. Med., vol. xxviii. p. 317. Rune- 
berg, Ibid., 1884, vol. xxxiv. pp. 1 and 266; and Berlin, klin. Wocb., 1897, No. 33. 
Citron, Ibid., 1897, p. 854 ; and Deutsch. Arch. f. klin. Med., vol. xlvi. Ranke, Mit- 
theil. a. d. rued. Klin. z. Wiirzburg, 1886, vol. ii. p. 189. 

Chemistry of Transudates. 

An idea of the chemical composition of the various forms of 
transudates may be formed from the following tables, taken from 
Hoppe-Seyler and Hammarsten, the figures corresponding to 1000 
parts by weight of fluid ; the specimens were taken from one 
individual : 



Pleura. Peritoneum. 



OZdema of 
the feet. 

Water . . 957.59 967.68 982.17 

Solids 42.41 32.32 17.83 

Albumin 27.82 16.11 3.64 

Ethereal extract 



Alcoholic extract 
Aqueous extract 
Inorganic salts 
Errors of analysis 



5.27 0.50 

f 3.71 

14.59 1Q94 I 1.10 

1U,y4 ] 9.00 

0.12 



Analysis of Hydkocele Fluid. 

Water 938.85 

Solids 61.15 

Fibrin (formed) 0.59 

Globulins . . m 13.52 

Serum-albumin 35.94 

Ethereal extract 4.02 

Soluble salts 8.60 

Insoluble salts 0.66 

Sodium chloride 6.19 

Sodium oxide 1.09 

Sugar and uric acid in small amounts are also, as a rule, found in 
transudates, and in one case of hepatic cirrhosis Moscatelli succeeded 
in demonstrating the presence of allantoin. v. Jaksch states that he 
has frequently been able to demonstrate the presence of urobilin in 
both transudates and serous exudates, even though red blood-cor- 
puscles and blood-coloring matter in solution were absent. Stich 



634 TRANSUDATES AND EXUDATES. 

also reports that in the ascitic fluid removed during life from a 
patient with hemorrhagic nephritis, urobilin was present. Peptone 
is never found ; and Pajikull states that nucleo-albumin is not 
present in transudates of non-inflammatory origin. Hammarsten, 
together with Pajikull, could, however, demonstrate an albuminous 
substance in transudates, which was regarded as a mucoid and which 
is present in exudates in small amounts only. It is rich in reducing 
substance and contains more nitrogen than the true mucins. 

Literature. — Moscatelli, Zeit. f. physiol. Chem., 1889, vol. xiii. p. 202. v. Jaksch, 
Zeit. f. Heilk,, 1891, vol. xi. p. 440. Eickhorst, Zeit. f. klin. Med., 1881, vol. iii, p. 
537. Stich, Munch, med. Wock., Oct. 29, 1901. 

Microscopical Examination of Transudates. 

Upon microscopical examination only a few isolated leucocytes 
and endothelial cells derived from the serous surfaces and under- 
going fatty degeneration are usually seen. Mast-cells and eosinophilic 
leucocytes have been observed in the ascitic fluid in cases of myeloge- 
nous leukaemia. Charcot-Leyden crystals were present at the same 
time. In cases in which the transudates have been confined for a 
long time plates of cholesterin are frequently found. They are 
especially abundant in hydrocele fluid. The technique which 
should be employed in the microscopical examination of transudates 
is described below. 

EXUDATES. 

Exudates may be serous, serofibrinous, hemorrhagic, seropurulent, 
purulent, putrid, chylous, or chyloid. Of these, the seropurulent, 
purulent, and putrid types are manifestly of inflammatory origin, 
while in the case of the serous, serofibrinous, and hemorrhagic 
forms it may at times be difficult to determine whether the fluid 
represents a transudate or whether it is an exudate. A detailed 
chemical and microscopical examination may then be necessary. 

Serous exudates are clear, of a light straw color, and present a 
specific gravity which usually exceeds 1.018. If blood-corpuscles 
are present in sufficient numbers to impart a distinct red color to 
the fluid, it is hemorrhagic ; the color may then vary from a light 
pink to a dark red. On standing, even the purely serous exudates 
generally undergo a certain degree of coagulation, which becomes 
more marked in the presence of blood ; exceptions, however, 
occur. Most important is the microscopical examination of the 
exudates. Generally speaking, the same methods are here employed 
as in the case of the blood, but the interpretation of the findings is 
not always easy. This is largely owing to the fact that the leuco- 
cytes often show evidence of degeneration, and that the fluid may 



EXUDATES. 635 

contain endothelial cells in addition to the morphological elements 
of the blood, which further increases the difficulties attending a 
proper classification (see Pus; page 641). The principal point at 
issue in the study of the cellular elements of exudates is the ques- 
tion as to the predominance of either lymphocytes or the polynu- 
clear elements of the blood. Widal and his school, more especially, 
have pointed out that whereas in exudates of non-tubercular, acute 
inflammatory origin the polynuclear neutrophilic leucocytes pre- 
dominate, the lymphocytes prevail in the chronic tubercular forms. 
His observations have been confirmed by numerous investigators, 
and the importance of cytodiagnosis in pleuritic effusions more 
especially is now well established. From the available data we 
may formulate the following conclusions. In the very earliest 
stages of tuberculosis involving the serous membranes there is found 
a variable number of neutrophilic leucocytes in addition to lympho- 
cytes and endothelial cells. Very soon, however, they diminish 
and in the later stages the lymphocyte is by far the predominat- 
ing cell, while the neutrophilic elements are present only in very 
small numbers. Generally speaking, the percentage of lymphocytes 
in tubercular pleurisies ranges from 50 to 98 per cent., increasing 
as the disease continues. 

In pleuritic effusions due to the pneumococcus and to streptococci 
during the serous stages, the neutrophilic leucocytes far outnumber 
the lymphocytes. In the pneumococcus cases, moreover, it is com- 
mon to meet with large numbers of endothelial cells, sometimes 
containing polynuclear leucocytes and red cells in their interior. 

In cases of traumatic and aseptic pleurisy, in association with 
diseases of the heart and kidneys, large endothelial cells, often pre- 
senting most grotesque appearances, occurring either singly or in 
groups of two, three, four, or more, are practically the only morpho- 
logical elements that are met with. 

French writers also describe a pleural eosinophilia, in which 
large numbers of eosinophilic cells — 6-54 per cent. — are found in 
the effusion, while in the circulating blood their number is not in- 
creased. Rabaut reports four cases of this kind. In one the effu- 
sion occurred secondarily in the course of syphilis ; in the second 
in a case of typhoid fever; the third was a case of phthisis, while in 
the fourth no diagnosis was made. 

Mast-cells are rarely seen in pleuritic effusions, and it has been 
observed that their granules are then quite readily soluble in water, 
so that they cannot be demonstrated with aqueous solutions of the 
usual dyes. Wolff notes a case in which the mast-cells constituted 
about 10 per cent, of the total number of leucocytes in a pleuritic 
exudate. 

Whether or not the conclusions which have been reached regard- 
ing the meaning of the prevalence of certain cell forms in pleural 



636 TRANSUDATES AND EXUDATES. 

effusions can be directly applied in the case of ascitic fluid remains 
to be seen. So far the subject has received but little attention. 
The same is true of the cytological study of joint-effusions. Widal 
reports that in three cases of acute rheumatism he found polynuclear 
leucocytes in the serous exudate, while they were absent in traumatic 
cases of arthritis. 

Of the cytological findings in the cerebrospinal fluid a detailed 
account will be given later (see page 653). 

Very important also is the study of the cellular elements which 
are found in serous exudates in cases of malignant disease of the 
serous membranes. Difficulty may here be encountered in the in- 
terpretation of the cellular findings, for on the one hand it is often 
difficult to distinguish the endothelial cells from leucocytes, as they 
take on phagocytic activity and often present the most bizarre 
forms. The nucleus which is normally centrally located takes up 
an excentric position, and enclosed within the cell we may find leu- 
cocytes and red cells. On the other hand, it is impossible by simple 
inspection to distinguish normal endothelial cells from cancer cells. 
In cases of doubt it is well to ascertain whether the epithelial ele- 
ments give the glycogen reaction and to hunt for the presence of 
mitosis. Quincke has pointed out that normal endothelial cells 
do not contain glycogen, and that a marked iodine reaction is very 
suggestive of carcinoma. Wolff, however, suggests that this test 
is probably not specific, and cites two instances in which he obtained 
a positive glycogen reaction, although a tumor did not exist. More 
important probably is the presence of mitoses. In non-malignant 
exudates epithelial cells never present evidence of mitosis, while in 
cases of tumor (sarcoma) they may.be found. Rieder regards their 
occurrence as pathognomonic of malignant disease. Commonly the 
mitosis is atypical ; the division of the nucleus is not followed by a 
division of the cell ; the chromosomes are short and show no polar 
or equatorial arrangement. 

In cases of neoplasm Quincke has also drawn attention to the 
occurrence of large numbers of fat droplets in the fluid, which may 
attain a diameter of from 40 to 50 //. At times, however, the fat 
droplets are so small and so numerous as to give a chylous appear- 
ance to the exudate. At other times a similar appearance is due to 
the presence of minute albuminous granules, which may be readily 
distinguished from fat by their insolubility in ether and the fact 
that they are not stained with the common fat dyes, such as 
Sudan, scarlet-R, and alkanin. The occurrence of numerous fatty 
acid crystals, arranged in groups, should also excite suspicion of a 
neoplasm. 

Should bits of tissue be obtained, a positive diagnosis of malig- 
nant disease may, of course, be made by the usual methods. Such 
particles should be placed at once in absolute alcohol or formalin. 



EXUDATES. 637 

Crystalline elements are not usually seen in serous or hemorrhagic 
exudates ; at times we meet with platelets of cholesterin. 

Technique. — In every case the fluid should be examined as soon 
after puncture as possible ; if this cannot be done at once, coagula- 
tion may be prevented by the addition of sodium citrate. The 
material is then placed in the ice box until a sediment has collected ; 
or this may be obtained at once by centrifugation, new portions of 
fluid being repeatedly used and the sediments combined. Cover- 
glass preparations may then be conveniently made, or smears on 
slides exactly as in the case of blood, care being taken to do as little 
injury to the cellular elements as possible. The smears should be 
very thin, so that the specimens will dry rapidly and but little 
chance is given for the cells to contract beyond their usual size. 
Subsequent treatment will depend upon the special points which are 
to be elicited. Unfortunately the leucocytes are often much changed, 
so that their classification may be attended by considerable diffi- 
culties. The polynuclear elements may appear mononuclear and 
not infrequently the neutrophilic granules can no longer be demon- 
strated (see page 641). For this reason the triacid stain is not to be 
recommended for routine work ; the eosinate is much better and 
will furnish as satisfactory results as can be obtained with a. pan- 
optic dye. Care should be had not to diagnose eosinophilia from 
the fact that cell granules are stained red, as the neutrophilic 
granules of degenerating cells are commonly amphophilic, viz., they 
stain both with acid and neutral dyes ; account must be taken of 
the size of the granules and the general structure of the cell. To 
differentiate pseudolymphocytes from true lymphocytes, Pappen- 
heim's methyl-green-pyronin may be employed, though it is not 
absolutely specific ; still it will be found that even though the pro- 
toplasm of other cellular elements may take the red color of the 
pyronin, the intensity is distinctly less than in the case of the 
lymphocytes proper. 

To study mitosis, hematoxylin and eosin may be employed, or 
the Romanowsky method in one of its various modifications. 

The glycogen reaction is demonstrated as in the case of the 
blood. 

Bacteriological Examination of Exudates. — In a measure the 
bacteriological examination of exudates has been supplanted by the 
cytological study, as outlined above ; especially as the bacteriological 
examination has been notoriously unsatisfactory in the most impor- 
tant group of effusions, viz., in those of tubercular origin. It is 
now 7 known that all exudates gradually become free from bacteria, 
even though at first they may have been caused by bacterial activity. 
As a result it is no longer justifiable to conclude that a process is 
tubercular because bacteriological examination of the exudate has 
given no positive result. If it is desired to cultivate organisms 



638 TRANSUDATES AND EXUDATES. 

that may be present, it is well to make a bouillon culture in every 
case so as to eliminate the bactericidal properties of the exudate as 
much as possible. In any event it is well to centrifugate the fluid 
in a sterile tube and to use the sediment for inoculations. The 
organisms which are most likely to be encountered are the pneumo- 
coccus, the various staphylococci, streptococci, and more rarely the 
colon bacillus and the typhoid bacillus. 

Literature.— Widal and Ravaut, "Cytodiagnostique des epanchements sero- 
fibi-ineux.de la plevre," Trans. XIII. Internat. Med. Cong. Paris, 1900. Barjou and 
Cade, " Etudes cytol.," etc., Arch. gen. d. med., August, 1902. Gulland, " Cytodiag- 
nosis," etc., Scott. Med. and Surg. Jour., June, 1902, p. 490. A. Wolff, "Transudates 
and Exudates," Zeit. f. klin. Med., 1902, vol. xxii. Hefte 5 u. 6. Quincke, Deutsch. 
Arch. f. klin. Med., 1882, vol. xxx. pp. 369 and 580. Eieder, Ibid., 1895, vol. liv. 
p. 544. 

Chemistry of Exudates. 

According to Moritz, an albumin is found in exudates that can 
be precipitated with acetic acid and which is absent in transudates. 
He regards this as serum-globulin which has undergone a change as 
a result of the inflammatory process. According to Matsumoto, on 
the other hand, the substance in question represents a mixture of 
fibrinoglobulin, euglobulin, and a small amount of pseudoglobulin ; 
in the filtrate, however, there is also some fibrinoglobulin (fibri- 
nogen) and euglobulin. He suggests that this last circumstance is 
probably referable to the small amount of salt in exudates and that 
in the first instance the pseudoglobulin is probably carried down 
mechanically. 

Still more recently Umber has studied the body in question and 
arrives at the conclusion that it belongs to the mucins. To its 
presence the mucinous character of such fluids is due. It is pre- 
cipitated by, the addition of acetic acid and is insoluble in an excess 
of the reagent unless the acid is present in great concentration. The 
body has markedly acid properties and is not coagulated by heat. 
It differs from the known mucins in the presence of a very small 
amount of reducing substance, which can only be demonstrated by 
special methods. It contains about 14 per cent, of nitrogen and no 
phosphorus. In neutral and feebly acid solution the substance does 
not coagulate (thus differing from globulins). The same body 
apparently was found by Salkowski in an exudate into the hip- 
joint. Umber calls this substance serosamucin. It amounts to less 
than 0.5 per cent. 

According to Umber and Stahelin, the serosamucin is essentially 
found in exudates referable to inflammatory processes or associated 
with new growths. In transudates, as Runeberg already pointed 
out, only a very slight turbidity results upon the addition of acetic 
acid, and not in all cases, moreover, so that a well-marked reaction 
viz., a marked precipitation upon the addition of acetic acid to the 
point of a distinctly acid reaction, may be regarded as a valuable 



EXUDATES. 639 

sign in the diagnosis between transudates and exudates. I append 
some of the results obtained by Umber : 

Ascites. 

No. of cases. Serosamucin. 

Hepatic cirrhosis . . 6 

Hepatic cirrhosis with chronic nephritis and phthisis 1 

Nephritis 1 

Mitral disease 3 

Pleural Exudates. 

Degeneratio cordis and nephritis 2 

Myocarditis 1 

Hepatic cirrhosis 1 

Lymphosarcoma (pleura intact post mortem) ... 1 

Carcinoma mammae with pleural metastases .... 1 -)- 

Tuberculosis of pleura 1 -j- 

Pleuritis exsudativa acuta 1 -j- 

Pleuritis and pericarditis 1 

In addition to the serosamucin and the common albumins men- 
tioned, some exudates may possibly also contain small amounts of a 
nucleo-albumin, as is suggested by the findings of Pajikull. Should 
ovarian cysts have ruptured into the peritoneal cavity, we may fur- 
ther find both pseudomucin and paramucin (which see). 

Isolation of Serosamucin. — To isolate the serosamucin, the follow- 
ing procedure may be employed (Umber) : The exudate is placed in 
the cold (about 0° C.) for a few hours in order to let the mor- 
phological elements settle down. The supernatant clear fluid is 
siphoned off, filtered, and diluted with an equal volume of water. 
Moderately dilute acetic acid is then added cautiously uutil the 
reaction is distinctly acid. The mucin separates out and settles at 
the bottom as a flocculent precipitate. It is washed with water con- 
taining a little acetic acid by decantation (two to three times) ; then 
with 80 per cent, alcohol and at intervals of twenty-four hours with 
stronger alcohol, and finally with ether. In this manner a prepara- 
tion is ultimately obtained which can be collected on a filter, while 
at first filtratiou is entirely out of the question as the substance 
rapidly clogs the pores of the filter. The isolated substance is prac- 
tically insoluble in water and with difficulty so in alkalies and fairly 
concentrated acids. 

Of the common albumins we meet with traces of fibrinogen and 
with fairly large amounts of globulins and true albumins (see page 
632). Their percentage may at times not appear so very large, but 
considering the large amount of fluid and the rapidity with which it 
may accumulate it is clear that the loss of nitrogen to the body in 
this form may be \ery considerable. Umber thus showed that in 
one of his cases 5000 grammes of albumin, representing about 
15,000 grammes of muscle tissue were lost within a year. 

Of interest further is the fact that Umber has also succeeded in 
demonstrating the existence of autolytic processes in exudates. He 



640 TRANSUDATES AND EXUDATES. 

found both albumoses and mono-amido acids, viz., leucin and 
tyrosin. Peptones were at no time encountered. 

Coriat has recently reported a case of polyneuritic delirium, in 
which pleurisy with effusion developed. In the effusion he could 
demonstrate a peculiar albuminous substance, which he regards as 
identical with Bence Jones albumin ; in the urine this substance 
could not be found. 

Literature. — Pajikull (Swedish ref. by Hammarsten : Jahresber. f. Thierchem., 
1893). Moritz, Munch, med. Woch., No. 42, 1902. Matsumoto, Deutsch. Arch., 1902, 
vol. lxxv. p. 409. Stahelln, Munch, med. Woch., 1902, No. 34. F. Umber, Zeitsch. f. 
klin. Med., 1903, vol. xlviii. p. 364. Coriat, " The Occurrence of the Bence Jones Albu- 
min in a Pleuritic Effusion," Am. Jour. Med. Sci., 1903, vol. cxxvi. p. 631. 

Pus. 

General Characteristics of Pus.— If pus, which usually pre- 
sents a color varying from yellowish gray to greenish yellow, is 
allowed to stand for a time, a liquid gradually appears at the top, 
and increases in amount until it is finally possible to distinguish 
two distinct layers, the one above — the pus-serum, the other at the 
bottom — the pus-corpuscles. Upon the number of the latter the 
consistence as well as the specific gravity of the pus is dependent. 
This may vary between 1.020 and 1.040, with an average of 1.031 
to 1.033. Fresh pus has always an alkaline reaction, which may 
become neutral or slightly acid upon standing, owing to the develop- 
ment of free fatty acids, glycerin-phosphoric acid, and lactic acid. 
The color of pus-serum may be a light straw, a greenish or a 
brownish yellow. 

Chemistry of Pus. — The chemical composition of pus-serum 
and pus-corpuscles may be seen from the following tables : 

Analysis of Pus-serum. 

I. II. 

Water 913.70 905.65 

Solids 86.30 94.35 

Albumins 63.23 77.21 

Lecithin 1.50 0.56 

Fat 0.26 0.29 

Cholesterin; 0.53 0.87 

Alcoholic extract . . . . 1.52 0.73 

Aqueous extract 11.53 6.92 

Inorganic salts 7.73 7.77 

Analysis of Pus-cokpuscles. 

I. II. 

Nuclein 342.37) 

Insoluble matter 205.66 \ 673.69 

Albumins 137.62 J 

kf thin } • - 143 - 83 {vaoo 

Cholesterin 74.00 72.83 

Cerebrin 51.991 mg4 

Extractives 44.33 / 



EXUDATES. 641 

Albumoses are usually present, and are derived from the pus-cor- 
puscles. Leucin and tyrosin are likewise frequently met with in the 
pus of old abscesses ; and fatty acids, urea, sugar, glycogen, biliary 
pigments and acids (in catarrhal jaundice), acetone, uric acid, xanthin- 
bases, cholesterin, etc., have occasionally been observed. 1 

Microscopical Examination of Pus. — Leucocytes. — If a drop 
of pus is examined with the microscope, it will be seen to contain 
innumerable leucocytes, many of which in perfectly fresh pus 
exhibit amoeboid movements. The cells in question are usually 
almost altogether of the neutrophilic variety, and it may be ques- 
tioned whether the lymphocytes ever occur in true pus. Even in 
cases of lymphatic leukaemia the predominating cell in abscesses is 
the poly nuclear leucocyte or its degeneration-forms. Mononuclear 
elements with basophilic protoplasm, however, are also met with, 
notably in the more chronic cases, but it is likely that they are 
derived from the connective-tissue cells and are not of hematogenic 
origin. Eosinophiles are only seen in pus under certain definite 
conditions, as in gonorrhoea (see below), and mast-cells also are 
quite uncommon. 

In pus that is not perfectly fresh it is usually not possible to dem- 
onstrate the presence of neutrophilic granules. In such cells, more- 
over, we commonly meet with degenerative changes aifecting the 
nuclei, such that the polymorphous nucleus in reality becomes poly- 
nuclear, while at the same time the individual fragments are dis- 
tinctly pyknotic. Such fragmentation was first noted by Ehrlich in 
a case of hemorrhagic smallpox and in various exudates, and has 
subsequently also been described by Michaelis and Wolff. The 
degeneration may proceed to a fragmentation of the entire cell, and 
it may then occur that specimens are met with in which the proto- 
plasm still contains neutrophilic granules (Ehrlich's pseudolympho- 
cytes, see page 83). On the other hand, a form of degeneration 
is seen in which the nucleus does not become pyknotic, but on the 
contrary swells to a large size and stains rather faintly with basic 
dyes. In such cells the protoplasm appears as a narrow rim and the 
impression is gained as though the cell were in reality a lymphocyte ; 
if at the same time the granules have been lost, the differentiation 
may indeed be impossible, unless transition-forms exist between the 
normal poly nuclear neutrophile and the type in question. 2 

Owing to resorption of water from accumulations of pus of long 
standing, such material finally assumes a caseous aspect, and the 
leucocytes will be seen to have greatly diminished in size, and to 
have assumed an angular, shrunken appearance ; it is then hardly 

1 M. Pickardt, " Z. Kenntniss d. Chemie path. Ergiisse," Berlin, klin. Woch., 1897, 
p. 844. 

2 L. Michaelis and A. Wolff, "Die Lymphocyten," Deutsch. med. Woch., 1901, vol. 
xxvii. p. 651. 

41 



642 TRANSUDATES AND EXUDATES. 

possible to demonstrate the presence of a nucleus, even after the 
addition of acetic acid. 

It is noteworthy that in cases of hepatic abscess referable to 
Amoeba coli it is seldom possible to demonstrate any normal leuco- 
cytes, and it will be seen that under such conditions the pus consists 
almost altogether of granular and fatty detritus, while in liver- 
abscesses due to other causes the leucocytes usually present a fairly 
normal appearance. 

Mast-cells are only exceptionally seen in pus. 

Giant Corpuscles. — So-called giant pus-corpuscles, measuring at 
times from 30 t a to 40 p. in diameter, have been observed in ab- 
scesses of the gum, hypopyon, and in the contents of suppurating 
ovarian cysts, but they do not appear to have any special significance. 
Upon careful examination these bodies will be seen to contain one 
oval nucleus, usually located eccentrically within the cell, and from 
one to thirty or even forty pus-corpuscles. 1 

Detritus. — Fatty and albuminous detritus in variable amount 
may be observed in every specimen of pus, and increases with the 
length of time it has been confined within the body. The same 
holds good for the presence of free nuclei, which were formerly re- 
garded as young pus-corpuscles, but which have now been definitely 
recognized as originating during the disintegration of the cor- 
puscles. 

Red Corpuscles. — Red blood-corpuscles in variable numbers are 
usually seen in every specimen, their appearance depending upon the 
length of time they have been confined. Pus-corpuscles may at 
times contain a red corpuscle. 

In doubtful cases it is always well to search carefully for the 
presence of tissue-elements, as only in this manner is it possible at 
times to recognize the character of the morbid process. As the data 
of importance have been detailed in other sections of this book 
(viz., Sputum and Urine), it is unnecessary to recapitulate at this 
place. 

Pathogenic Vegetable Parasites. — Of the pathogenic organisms 
which are of especial interest from a clinical standpoint may be 
mentioned the true pus-organisms, notably the Staphylococcus 
pyogenes aureus and the Streptococcus pyogenes ; furthermore, 
the tubercle bacillus, the Actinomyces hominis, the bacillus of 
glanders, the bacillus of anthrax, leprosy, tetanus, influenza, and 
FrankePs pneumococcus, etc. The majority of these have already 
been described, and the reader is referred for more detailed informa- 
tion to special works on bacteriology. In this connection it will 
suffice to state that, so far as pleural exudates are concerned, an 
absence of micro-organisms is usually indicative of tuberculosis, 
while the presence of FrankePs pneumococcus in exudates forming 

1 BotioLicr, Vircbow's Archiv, 1SG7, vol. xxxix. p. 512. Bizzozero, loc. cit. 



EXUDATES. 643 

in the course of a pneumonia appears to be a favorable omen as 
regards the origin of the pleuritic effusion. 1 

Protozoa, with the exception of the Amoeba eoli, have only rarely 
been found. Kiinstler and Pitres 2 observed numerous large spores 
with from ten to twenty crescentic corpuscles in pus taken from the 
pleural cavity of a man, which closely resembled the coccidia of 
mice. Litten 3 observed cercomonads in fluid withdrawn from a 
pleural cavity. Trichomonads have been found in empyema in con- 
nection with pulmonary gangrene. 

Most important in this connection is the demonstration of the 
Amoeba coli in the pus, and in cases of liver-abscess an examination 
with this view should never be neglected, as the prognosis will to a 
large extent depend upon the results obtained. So far as the occur- 
rence of amoebae in pus is concerned, the observation of Flexner, 
who demonstrated their presence in an abscess of the lower jaw, 
shows that they should not be looked for in the pus of abscesses of 
the liver or lung only. 

Vermes. — Of these, the filaria and hydatids are rarely observed 
in this country. Bothriocephalus leguloides has been found in the 
pleural cavity of a Chinese patient. 

Crystals. — As has been stated, crystals of cholesterin are fre- 
quently found in old pus and in exudates of long standing, but are 
rarely seen in recent exudates. They may be recognized by their 
characteristic form and their chemical reactions, as described in the 
chapter on the Feces (page 282). Triple phosphates, fatty acid 
crystals, and hseinatoidin are likewise frequently seen, the presence 
of the latter, of course, indicating a previous admixture of blood. 

The technique to be employed in the examination of pus is as a 
rule simple. Cover-glass preparations or smears on slides are pre- 
pared as in the case of the blood and are then stained according to 
the points that are to be elicited. For routine work, the eosinate 
of methylene-blue will be found very useful. If the pus corpuscles 
are still fairly fresh, the neutrophilic granules are readily stained ; 
it will be noted, however, that very commonly they exhibit a more 
decided red, which is referable to certain degenerative changes which 
cause the granules to assume an affinity for acid dyes as well. Bac- 
teria that may be present are usually well shown. If the pus is 
older and the cells have lost their granules, Pappenheim's pyronin- 
m ethyl-green will be found of value in the study of the mononuclear 
forms (see also page 136). 

1 Ludwig Ferdinand v. Bayern, Arch. f. klin. Med., 1892, vol. 1. p. 1. Frankel, 
Charite Annal., 1SS8, vol. xiii. p. 3 47. 

2 Kiinstler u. Pitres, Conrpt. rend, de la Soc. de Biol., 1884, p. 523. 

3 Litten, Verhandl. d. Cong. f. inn. Med., 1886, vol. v. p. 417. 



644 TRANSUDATES AND EXUDATES. 

Gonorrhoeal Pus. 

In the very earliest stages of the disease the pus contains large 
numbers of eosinophilic cells besides the common polynuclear neu- 
trophiles. But at the same time and throughout the course of the 
disease mononuclear non-granular elements, with basophilic proto- 
plasm, are also seen. The larger number of the latter are of the type 
of the large mononuclear leucocyte and transition-form of Ehrlich, 
but a certain percentage is also represented by the lymphocytes, both 
of the small and the large variety. 

The neutrophilic elements in gonorrhoeal pus commonly present 
evidence of degeneration. In some a loss of granular material has 
manifestly taken place, and it can be demonstrated that in most of 
the cells the granules are no longer absolutely neutrophilic, but 
have become amphophilic — that is, from a neutral mixture they 
take up the neutral dye, but they can also be stained with, acid dyes. 
With the triglycerin mixture, for example, they are stained red by 
the eosin. 

As to the eosinophiles, O wings, who has studied this problem in 
my laboratory, came to the following conclusions : 

1. Eosinophilic leucocytes are present in gonorrhoeal pus in a 
large percentage of cases. They may be absent, however, even 
when a marked hyperleucocytosis and eosinophilia exist in the 
blood. 

2. Their number varies pari passu with the number present in 
the blood, and the percentage in the pus is never in excess of the 
percentage in the blood. 

3. Gonococci are rarely found in eosinophilic leucocytes. 
Mast-cells may also occur in gonorrhoeal pus ; a remarkable case 

is reported by Neisser, in which the pus consisted practically exclu- 
sively of such elements. 

As regards the distribution of gonococci in the different cellular 
elements, it is noteworthy that they are principally found in the poly- 
nuclear neutrophiles, while they are less commonly seen in the mono- 
nuclear leucocytes and transition-forms. In the small lymphocytes 
they are not encountered, and it is uncommon to find them in the 
eosinophilic cells. 

Generally speaking numerous gonococci, eosinophiles, and a small 
number of lymphocytes are found in cases of recent gonorrhoea, 
while during an exacerbation of a chronic process only a few cocci 
and numerous mononuclear elements are encountered. 

The gonococcus (Neisser) (Plate XXII.) occurs in the form of 
small oval or coflFee-bean-shaped granules, grouped in twos and fours 
resembling a German biscuit ; the individual cocci measure about 
1.25 ju in length by 0.7 fi in diameter. As a rule they are found 
enclosed within pus-corpuscles and epithelial cells ; but they may 



EXUDATES. 645 

also occur free in the pus obtained from the urethra, in the vaginal 
discharge, and more rarely in urinary sediments as in cases of com- 
plicating prostatitis, peri -urethritis, etc. In cover-glass specimens 
account should be taken only of those organisms which are enclosed 
within cellular elements, as these alone may be regarded as charac- 
teristic. To this end a drop of the discharge is spread in a thin 
layer upon a slide or a cover-glass, dried in the air, and fixed by 
passing three or four times through the flame of a Bunsen burner. 
The specimens may then be stained with any one of the basic anilin 
dyes. In my laboratory the eosinate of methylene-blue is almost 
exclusively used for this purpose (see page 123). The organisms 
are thus colored blue, while the granules of eosinophilic leucocytes, 
which may be present at the same time, appear a bright red or a 
brownish red. After five minutes the excess of stain is washed off, 
the preparations are rinsed in water, dried with filter-paper, and 
examined with a high power. 

The gonococcus is decolorized by Gram's method and can in this 
manner be distinguished from similar organisms that may be present. 
Of the four kinds of diplococci which may be found in urethritis 
besides the gonococcus, only two forms are similarly decolorized, and 
these two are only rarely seen. "We may conclude that in 95 per 
cent, of all cases Gram's method permits a definite conclusion as to 
the presence or absence of the true organism. Gram's method is 
here best employed in the modification suggested by Weinrich : 
The preparations are fixed by drawing through the flame of a Bun- 
sen burner and are then stained for from one to two minutes in 
FrankePs carbol-gentian-violet solution (10 parts of a saturated alco- 
holic solution of gentian-violet to 90 parts of a 25 per cent, solution 
of carbolic acid). Without washing they are placed for one to three 
minutes in LugoPs solution (1 gramme of iodine, 2 grammes of 
potassium iodide, and 300 c.c. of distilled water), and again without 
washing in absolute alcohol, until the alcohol ceases to extract color 
(about one and one-half minutes) ; they are now washed in water, 
counterstained with Bismark-brown, washed, dried, and mounted. 
The Bismark-brown solution is prepared as follows ; 3 grammes 
of the dye are dissolved in 70 c.c. of hot water; 30 c.c. of 96 per 
cent, alcohol are added ; the mixture is well stirred and filtered. 

Of special interest is the observation of Unna and Plato, that the 
gonococcus can be stained in the living leucocyte with Ehrlich's 
neutral red. The method employed is simple. A small drop of the 
fresh pus is mixed with an ose of a dilute solution of neutral red in 
normal salt solution (1 c.c. of a saturated aqueous solution to 100 
c.c), and examined either as hanging drop or mounted on a slide as 
usual. Thus prepared, a certain number of the intracellular gono- 
cocci are stained a deep red, while others are not stained ; and it 
may be observed on warming the slide, so as to elicit amoeboid 



646 TRANSUDATES AND EXUDATES. 

movements, that some of the gonococci which are stained so long as 
they remain within the granular portion of the leucocytes, are gradu- 
ally decolorized when they come to lie in the homogeneous ectosarc, 
and are colored again on returning to the granular endosarc. Plato 
states that he has examined numerous other intracellular organisms, 
including pseudogonococci, but that he has never observed as rapid 
and intense staining as with the true gonococci. He therefore sug- 
gests that with neutral red it may be possible to differentiate the true 
gonococcus from pseudogonococci. Extracellular gonococci, as well 
as numerous other bacteria, are not stained even after an exposure 
of several days. 

The organism grows best on hydrocele agar (see below). The 
surface colonies are pale, grayish, translucent and finely granular, 
with finely notched borders. In bouillon and blood-serum mixed it 
forms a membrane, while the fluid remains clear. 

When no discharge can be obtained from the urethra, or an ex- 
amination of such discharge is negative, positive results may at times 
still be obtained if some of the gonorrhoea! threads are examined 
which may be found floating in the urine. In these the organisms 
can occasionally be demonstrated after months and even years have 
elapsed after primary infection. 

Preparation of Hydrocele Agar (Gushing). — The fluid 
(hydrocele or ascitic) is obtained sterile, the locality of puncture being 
carefully sterilized by modern surgical methods, the sterile trocar cov- 
ered at its external end with sterile gauze, so as not to be infected by 
the operator's hand, and the fluid collected in sterile flasks, the sterile 
stoppers being then replaced. When collected in this way it rarely 
becomes contaminated and may often be kept for months before using. 
This fluid is mixed with ordinary nutrient agar. A number of com- 
mon agar slants are placed in the autoclave for five minutes. This 
liquefies the agar and at the same time thoroughly sterilizes the tubes 
and cotton stoppers. The slants are then put in a water-bath at 55° 
C, so as not to coagulate the albumin when mixed with the agar. 
The stopper having been removed from a small flask of hydrocele 
fluid, the top of the flask is flamed and the albuminous fluid then 
poured into an agar tube (the top of which has also been flamed) in 
the proportion of a little more than one to two. It is well to have 
as much of the hydrocele fluid as the future solidity of the medium 
will allow. Ordinary agar will allow not quite equal parts of the 
two. The stopper is then returned to the agar tube, which is imme- 
diately slanted. On these slants the gonococci grow most abun- 
dantly in or near the liquid which is squeezed out of the medium 
and collects at the bottom of the tube. Some cultures will main- 
tain a vigorous growth after numerous transplantations, while others 
again grow only two or three times, or indeed once only. 



EXUDATES. 647 

Literature.— Janowski, Arch, f, exper. Pathol., 1895, vol. xxxvi. p. 15. L 
Michaelis and A. Wolff, " Die Lymphocyten," Deutsch. rned. Woch., 1901, vol. xxvii. p. 
651. A. Pappenheim, Virchow's Archiv, 1901, vol. clxix. p. 72. Neisser, Centralbl. f. 
d. med. Wiss., 1879, vol. xvii. p. 497. J. Plato, " Ueber Gonokokkenfarbung mit Neu- 
tralroth," etc., Berlin, klin. Woch., 1899, p. 1085. E. E. Owings, "The Infectiousness 
of Chronic Urethritis," Bull. Johns Hopkins Hosp., 1897, p. 210. H. H. Young, 
Welch Festschrift, Johns Hopkins Press., 1900, p. 677. 

Putrid Exudates. 

Putrid exudates are observed following perforation of a gangren- 
ous focus or of a gastric or intestinal ulcer into one of the body- 
cavities. At other times they are encountered in cases of neoplasm, 
and at times even without apparent cause. The material obtained 
in such cases has a brown or brownish-green color, and emits an 
odor which in itself indicates the character of the exudate. Micro- 
scopically, cholesterin, hsematoidin, and fatty acid crystals, as well 
as degenerating leucocytes, are found. In cases in which aspiration 
of a higher intercostal space reveals the presence of serous fluid, 
while putrid material is obtained at a lower point, the existence of a 
subphrenic abscess should be suspected. In such cases a pure cult- 
ure of the Bacillus coli communis has been obtained. The reaction 
of putrid exudates is usually alkaline, but an acid reaction may be 
obtained in cases of perforation of a gastric ulcer ; the Sarcina ven- 
triculi and saccharomyces may then also be found. 

Chylous and Chyloid Exudates. 

Chylous and chyloid exudates have been repeatedly observed. 
They are most frequently met with in the abdominal cavity (one 
hundred and four times out of the total number of one hundred and 
fifty-five, which have thus far been reported), less commonly in the 
pleural cavity (forty-nine times), and only rarely in the pericardial 
sac (twice only). Quincke believes that the two forms can be 
etiologically distinguished from one another by means of a micro- 
scopical examination, as the cloudy appearance in the chyloid form 
is usually referable to the presence of endothelial or epithelioid cells 
undergoing fatty degeneration. Later observations, however, have 
shown that the differentiation of the two forms cannot be made upon 
this basis, as the same anatomical lesion, such as carcinoma, may at 
times give rise to the formation of a chylous exudate, at others to 
that of the chyloid form, and both, moreover, may coexist. 

Senator claimed that the presence of more than mere traces of 
sugar is strongly suggestive of the chylous nature of the exudate. 
Possibly this observation may be of some value, but it must not be 
forgotten that sugar is commonly met with in all forms of trans- 
udates and exudates. Only the presence of more than 0.2 per cent, 
is of value. 

Chylous exudates in their general appearance resemble milk, while 



648 TRANSUDATES AND EXUDATES. 

chyloid fluid is more suggestive of pus. The turbity in both cases 
is usually referable to the presence of innumerable fat-globules, 
which are especially abundant in the chylous form. In chyloid 
exudates the origin of the fat from cellular elements is often appar- 
ent at once ; but, as has been said, it is impossible to draw definite 
etiological conclusions "from that difference. Some chyloid exudates 
contain no fat at all, and Lion has shown that the milky appearance 
in such cases is owing to the presence of a curious albuminous 
substance, belonging to the class of nucleo-albumins. Bernert, on 
the other hand, claims that the substance in question belongs to the 
globulins, and is closely associated with certain lecithins. 

Literature. — Quincke, loc. cit. Boulengier, Schmidt's Jahrb., 1890, vol. ccxxvi. 
p. 28. 



CHAPTER IX. 
THE CEREBROSPINAL FLUID. 

According to our present knowledge, the cerebrospinal fluid is 
secreted by the choroid plexuses into the lateral ventricles. Passing 
through the foramina of Monro, the third ventricle, and the aque- 
duct of Sylvius, on the one hand, it reaches the fourth ventricle and 
enters the cistern-like subarachnoid spaces at the base of the brain, 
through the foramen of Magendie and the lateral clefts of the fourth 
ventricle. On the other hand, a certain portion of the fluid reaches 
the same destination directly through the cleft in the descending horn 
of each lateral ventricle. The larger portion of the fluid then passes 
upward through the subarachnoid spaces along the convexity of the 
brain to the Pacchionian granulations, while the smaller portion 
enters the vertebral canal through the subarachnoid spaces of the 
spinal arachnoid membrane. 

Within recent years puncture of the vertebral canal has been 
frequently resorted to, both for therapeutic and diagnostic purposes. 
The practical value of this method of diagnosis is now beyond ques- 
tion, and it is to be hoped that ere long physicians will resort to 
spinal puncture in obscure cases of cerebrospinal disease with as 
little hesitancy as puncture of the thoracic and abdominal cavities is 
now practised. 1 

The operative method to be employed is the following : with the 
patient placed upon his left side, — some observers prefer the sitting 
posture, — and the body bent well forward, a long aspirating-needle 
is introduced upon a level with the lower third of the third or fourth 
lumbar spinous process, and about 1 cm. to the side of the median 
line, the needle being directed slightly upward and inward. The 
depth to which it is necessary to puncture will, of course, vary with 
the age of the patient. In a child two years of age the vertebral 
canal may be reached at a depth of 2 cm., while in the adult it is 
necessary to insert the needle for a distance of from 4 to 8 cm. As 
soon as the subarachnoid space is reached cerebrospinal fluid will 
flow from the needle. Aspiration should always be avoided. 

Some writers have advised that the operation be performed under 

1 H. Quincke, Verhandl. d. X. Cong. f. inn. Med., 1891. A. Hand, " A Critical 
Summary of the Literature on the Diagnostic and Therapeutic Value of Lumbar 
Puncture," Am. Jour. Med. Sci., 1900, vol. cxx. p. 463. A. Stadelmann, " Klinische 
Erfahrungen mit d. Lumbal puucti on," Deutsch. nied. Woch., 1897, p. 745. 

649 



650 THE CEREBROSPINAL FLUID. 

narcosis ; and without doubt this may be necessary at times, particu- 
larly when contracture of the dorsal muscles exists. In the majority 
of cases, however, it is not necessary. 

Amount. — So far as I have been able to ascertain, no observations 
have been made regarding the amount of fluid which may be obtained 
by puncture in normal individuals. In all probability, however, this 
is small. Under pathological conditions the amount may vary from 
a few drops to 100 c.c, and even more. In general terms it may 
be stated that the amount is directly proportionate to the degree of 
intracranial pressure. Exceptions, however, are frequent. Small 
amounts of cerebrospinal fluid or none at all may thus be obtained 
when owing to the formation of a thick exudate or the existence of 
a cerebral tumor communication between the basilar subarachnoid 
spaces of the brain and those of the spinal cord has been interrupted. 
Whenever, then, symptoms of intracranial pressure exist, while no 
fluid or minimal amounts only can be obtained by puncture, the 
conclusion will usually be justifiable that we are dealing with a 
purulent meningitis or with a tumor of the brain, and more especially 
of the cerebellum. It should be remembered, however, that the 
same result may be obtained in cases of obliteration of the aqueduct 
of Sylvius, or when sclerotic processes involve the foramen of 
Magendie, which is occasionally observed in certain forms of hydro- 
cephalus. Adhesions of the pia mater to the arachnoid and the 
dura mater may, by interfering with the flow of cerebrospinal fluid, 
also lead to the formation of hydrocephalus, but in these cases a 
tumor can usually be excluded, as the changes in question always 
develop as sequelae to a meningitis. A serous or tubercular menin- 
gitis, as well as acute hydrocephalus and tetanus, can, however, 
always be excluded when only minimal amounts of fluid are obtained 
by puncture. The largest amounts, on the other hand, are seen in 
cases of serous meningitis, tubercular meningitis, and cerebral tumors, 
which do not interfere with the circulation of the cerebrospinal 
fluid. In epilepsy Pellagrini usually obtained amounts varying 
between 10 and 15 c.c. 1 

Appearance. — Normal cerebrospinal fluid, as well as that obtained 
in cases of serous meningitis, tubercular meningitis, hydrocephalus, 
and tumors of the brain, is perfectly clear, and as a rule colorless 
unless a small blood-vessel has been punctured, when the fluid may 
present a slightly reddish tinge. More or less pronounced yellow 
shades are, however, at times observed. Important from the stand- 
point of diagnosis is the fact that in cases of hemorrhage into the 
ventricles pure blood is obtained, while such a result is, of course, a 
mechanical impossibility in cases of epidural hematoma. In subdural 
hematoma, on the other hand, blood may also find its way into the 
subarachnoid space, but the amount is always small, and cannot be 

1 Pellagrini, La Eiforma nied., 1901, Ann. 17, vol. ii. p. 638. 



SPECIFIC GRAVITY. 651 

compared with that seen in cases of ventricular hemorrhage. When- 
ever, then, as in traumatic cases with severe cerebral symptoms, the 
surgeon is confronted with the question whether or not to trephine, 
puncture of the subarachnoid space may furnish much valuable 
information. If in such cases no blood at all is found, it may be 
inferred that an epidural hsematoma or a subdural hematoma of 
slight extent only exists ; an operation may then be performed. If, 
however, pure blood is encountered, it would be justifiable to assume 
the existence of extensive injury to the brain-substance proper, 
or, in cases in which the history is obscure, an intracerebral hem- 
orrhage with rupture into the ventricles. In such cases the idea 
of an operation would, of course, be entertained only under excep- 
tional conditions. If, further, the fluid is only tinged with blood, 
a subdural hsematoma probably exists, and an operation should 
be advised. Accidental hemorrhage, viz., hemorrhage referable to 
the puncture itself, can be readily recognized, as the first few drops 
only are then tinged with blood, or the blood appears only after the 
flow has been definitely established ; the amount, moreover, is insig- 
nificant. 

Cloudy fluid is obtained in all cases of purulent meningitis unless 
the disease is limited to a very small area. This is, of course, most 
important from a diagnostic standpoint. Cases of abscess of the brain 
or sinus thrombosis occur again and again in which the question as 
to the advisability of operative interference is largely dependent 
upon the presence or absence of a complicating purulent meningitis. 
In certain instances a satisfactory conclusion may, of course, be 
reached without puncture ; but in many others this is impossible, 
and Lichtheim's dictum, that an operation should never be under- 
taken in such cases unless the integrity of the meninges has been 
established by spinal puncture, should be borne in mind. 

The degree of cloudiness naturally varies in different cases, and 
while in some instances the character of the fluid is seropurulent, 
pure, creamy pus may be found in others. Generally speaking, a 
cloudy fluid indicates the existence of an acute inflammatory process 
or an exacerbation of a chronic process. 

Important, furthermore, is the fact that the fluid in non-inflam- 
matory diseases of the brain, such as tumor or abscess, rarely 
undergoes coagulation, while this is the rule in all inflammatory 
diseases. In tubercular meningitis the coagula are very delicate, 
and may be well compared to spider-webs extending throughout 
the fluid, while in purulent meningitis the coagula are much firmer. 

Specific Gravity. — The specific gravity of cerebrospinal fluid 
normally varies between 1.005 and 1.007, corresponding to the 
presence of from 10 to 15 pro mille of solids. Under pathological 
conditions variations from 1.003 to 1.012 may be observed, the 
specific gravity, generally speaking, being higher in the inflamma- 



652 THE CEREBROSPINAL FLUID. 

tory than in the non-inflammatory diseases of the brain. From a 
diagnostic standpoint, however, determination of the specific gravity 
is of little value, as numerous exceptions to the above rule occur. 

The reaction is always alkaline. 

Chemical Composition. — An idea of the chemical composition of 
the cerebrospinal fluid may be formed from the following analyses, 
taken from Gautier and Zdarek : 

Water 987.00 

Albumin 1.10 

Fat . . 0.09 

Cholesterin 0.21 

Alcoholic and aqueous extract, minus salts \ ~ ^r 

Sodium lactate / 

Chlorides 6.14 

Earthy phosphates 0.10 

Sulphates 0.20 

Zdarek 1 $ Analysis. 

Water 989.54 

Solids . 10.45 

Organic solids 2.09 

Mineral ash 8.35 

Albumins 0.76 

Ethereal residue . . 0.35 

Aqueous residue 8.22 

Sulphuric acid (S0 3 ) 0.04 

Chlorine " 4.24 

Carbon dioxide 0.49 

Potassium oxide 0.16 

Sodium oxide 4.29 

Mineral ash, insoluble in water 0.16 

Glucose 0.10 

In addition, urea is at times found, as also a substance which 
reduces Fehling's solution and gives rise to a brown color when 
boiled with caustic potash, but which neither undergoes fermentation 
nor forms an osazon when treated with phenylhydrazin. The sub- 
stance in question is generally regarded as pyrocatechin. Its amount 
varies between 0.002 and 0.116 per cent. According to C. Ber- 
nard, glucose may also be present, but it is questionable whether 
this is the case under normal conditions (see below). Nawratzki 
discovered a reducing substance in his cases, which was demon- 
strated to be glucose ; his subjects, however, were unfortunately not 
normal, but general paretics with fever. Pyrocatechin was absent. 
Zdarek T reports a recent case of anterior meningocele in an otherwise 
normal individual in which the fluid reduced Fehling's solution and 
gave a glucosazon with phenylhydrazin. The substance in question 
was dextrorotatory, the amount corresponding to 0.1 per cent, of 
glucose. 

So far as the albuminous bodies are concerned which may be found 

1 E. Zdarek, Zeit. f. phys. Chem., 1902, vol. xxxv. p. 202. 



MICROSCOPICAL EXAMINATION. 653 

in the cerebrospinal fluid, serum-albumin is said to be present only 
under exceptional conditions, while normally a mixture of globulin 
and albumoses is found. The question whether or not mucin may 
also be present is still undecided. 1 

Under pathological Conditions the amount of albumin may vary 
considerably, and is of diagnostic importance. According to the 
majority of observers, the figure given in the above analysis is 
too high, and it is doubtful whether 1 pro mille may be regarded as 
normal. The lowest values have been obtained in cases of chronic 
hydrocephalus (traces only), meningitis serosa (0.5 to 0.75 pro mille), 
and tumors of the brain (traces to 0.8 pro mille) ; while the largest 
amounts have been found in chronic hydrocephalus the result of 
hypersemia (1 to 7 pro mille), and in tubercular meningitis (1 to 3 
pro mille). Nawratzki in recent examinations found amounts varying 
between 0.047 and 0.170 per cent., but the subjects of his investi- 
gation had fever at the time. 

Lichtheim claims to have found glucose — by means of the phenyl- 
hydrazin test — in all cases of tumor which he examined. In cases 
of tubercular meningitis, on the other hand, a positive result was 
only exceptionally obtained. Quincke also reports that he was able 
to demonstrate the presence of sugar whenever the liquid obtained 
was sufficient in amount for the necessary tests. Unfortunately, 
however, he does not detail his cases. Concetti found no sugar in 
hydrocephalic fluid. 

The experience of other observers does not agree with that of 
Lichtheim and Quincke ; and Fiirbringer, 1 who has thus far reported 
the largest number of spinal punctures, found sugar in only two 
cases of diabetes associated with tuberculosis. 

According to Gumprecht, the normal cerebrospinal fluid also con- 
tains traces of cholin. 

Microscopical Examination. — According to Widal, Sicard, 
Ravaut, and others, it is possible to make a diagnosis of the character 
of the morbid process in cases of meningitis from the morphology of 
the cells contained in the cerebrospinal fluid. Generally speaking, 
lymphocytes prevail if the inflammatory process is tubercular in 
origin, while the polymorphonuclear variety predominates in the 
non-tubercular cases. Exceptions to this rule, however, occur. 
Marcou-Mutzner 3 thus reports a case in which the prevailing cell 
was a polymorphonuclear leucocyte, notwithstanding the fact that, 
as autopsy showed, there existed a typical pulmonary and meningeal 
tuberculosis. He also cites a case, reported by Rendu, in which 
lymphocytes predominated, and in which autopsy showed fracture 
of the base of the skull. 

1 Stadelmann, Mitth. a. d. Grenzgebiete d. Med. u. Chir., vol. ii. Comba. Clin, med., 
1899 (cited in Arch. d. Med. d. Enfants, 1900). Lenhartz, Verhandl. d. XIV. Cong, 
f. inn. Med., 1900. 

2 Fiirbringer, Verbandl. d. XV. Cong. f. inn. Med., 1901, 

3 Marcou-Mutzner, Arcb. gen. d. Med., Sept., 1901, p. 345. 



654 THE CEREBROSPINAL FLUID. 

Bendix reports 8 cases of meningitis from Minkowsky's clinic in 
which an examination was made in this direction. Five of these 
were cases of meningeal tuberculosis, and in all the cellular elements 
were mostly lymphocytes, while the poly nuclear cells were present 
in only small numbers. The 3 remaining cases were due to the 
meningococcus intracellularis. In 2 of these the prevailing cell was 
the polynuclear neutrophile, but in 1 the lymphocytes were in excess. 
Bendix suggests that possibly the duration of the disease rather 
than its bacteriological character may be the deciding factor in 
determining the morphological findings ; that in chronic cases the 
lymphocytes prevail, which would account for the exception, as in 
this case the disease had extended over several months. 

It thus appears that the cytological examination of the cerebro- 
spinal fluid does not furnish information which is practically con- 
clusive as in the case of pleuritic exudates (which see). Nevertheless 
the exceptional cases are comparatively few, and in all doubtful 
cases a careful study of the cellular elements is demanded. 

The technique to be employed in the cytological study of the 
cerebrospinal fluid is the same as in the case of pleural exudates 
(see page 637). 

Very important also from a diagnostic point is the fact that patho- 
genic micro-organisms may be found. Lichtheim, Fiirbringer, Frey- 
han, Dennig, and Frankel were thus able to demonstrate the presence 
of tubercle bacilli in a fairly large number of cases of tubercular 
meningitis. Other observers, it is true, have been less fortunate, 
but the fact that Fiirbringer found tubercle bacilli in thirty cases out 
of thirty-seven is certainly significant. Schwarz states that he ob- 
tained positive results in sixteen out of twenty-two cases, and 
Slawyk and Manicatide found bacilli in all of nineteen cases (six- 
teen times by direct microscopical examination, and three times by 
the animal experiment). In order to examine for tubercle bacilli, 
the fluid should be placed on ice for from six to twenty-four hours, 
until a slight coagulum has formed, when the fine, spider-web-like 
threads of fibrin are transferred to a cover-slip, spread in as thin a 
layer as possible, and stained as described in the chapter on the 
Sputum. If a centrifugal machine is available, the examination 
may, of course, be made at once ; the chances of finding the bacilli 
are then also much greater. In every case a large number of speci- 
mens should be prepared before the search is abandoned. Only a 
positive result, however, is of value, and in doubtful cases recourse 
should be had to the animal experiment. 2 

In the diagnosis of epidemic cerebrospinal meningitis lumbar 
puncture is of signal value, as the Diplococcus meningitidis intracellu- 
laris of Weiehselbaum-Jager can be demonstrated in a large per- 

1 Fiirbringer, loc. cit, Wentworth, Arch, of Pediat., Nov., 1899. 



MICROSCOPICAL EXAMINATION. 655 

centage of cases. Councilman thus states that during a recent 
epidemic of the disease in Boston lumbar puncture was performed 
in fifty-five cases, and that in the fluid obtained the diplococci were 
found on microscopical examination or in culture in thirty-eight 
cases. The average time from the onset of the disease before spinal 
puncture was made was seven days in the positive cases, and seven- 
teen days in the negative cases. The longest time after the onset 
in which a positive result was obtained was twenty -nine days. 
Similar results have also been reached by other observers. 

The organism in question is a diplococcus, each half being of 
about the same size as the ordinary pathogenic micrococci. It is 
readily stained with the usual dyes, and decolorized by Gram's 
method. Short chains of from four to six and tetrads may at times 
be seen. It grows best upon Loffler's blood-serum mixture, form- 
ing round, whitish, shining, viscid-looking colonies, with smooth, 
sharply defined outlines, which may attain a diameter of from 1 to 
1J mm. in twenty-four hours. Their cultivation upon plain agar, 
glycerin-agar, and in bouillon is less reliable. 

In order to obtain the best results, it is necessary to use large 
amounts of the exudate, and to make a number of cultures, as 
many of the organisms are usually dead, or at least will not grow. 
In ordinary cover-slip preparations they are often numerous, and 
are found enclosed in the polynuclear leucocytes. Their number 
then varies considerably. On the one hand, only one or two may 
be present in a cell, while in others they may be so closely packed 
as to obscure the nucleus. 

Mixed infections are not uncommon in epidemic cerebrospinal 
meningitis. Councilman thus found the pneumococcus in seven 
cases, and Friedlander's bacillus in one. Terminal infections with 
staphylococci and streptococci also occur. 

In other forms of purulent meningitis a large variety of organisms 
has been found. Wolf gives the following figures, resulting from an 
analysis of 174 cases, in which epidemic cerebrospinal meningitis is, 
however, included : in 44.23 per cent, the pneumococcus was found ; 
in 34.48 per cent, the Diplococcus meningitidis intracellularis ; in 
3.45 per cent, staphylococci; in 8.03 per cent, streptococci, in 1.13 
per cent, the bacillus of Friedlander ; in 2.87 per cent, the Bacillus 
typhosus; in 1.72 per. cent, the bacillus of Neumann-Schaffer, and 
in 2.87 per cent, the Bacillus coli communis, the Bacillus pyogenes 
foetidus, the Bacillus aerogenes meningitidis, and the Bacillus mallei, 
while no bacteria were found in 1.15 per cent, of the cases. In 
two cases Pfeiffer's influenza bacillus has also been encountered in 
the cerebrospinal fluid during life. 

In the African sleeping sickness trypanosomes are commonly 
found in the cerebrospinal fluid, obtained by lumbar puncture. 
Castellani obtained the organism in twenty cases of thirty-four, and 
Bruce found it in all of thirty-eight cases (see page 191). 



656 THE CEREBROSPINAL FLUID. 

Toxicity. — While normal cerebrospinal fluid possesses distinct 
toxic properties, it has been found that in disease the toxicity may 
be markedly increased. Bellisari has thus shown that the fluid of 
individuals suffering from general paresis is more toxic than that 
of normal individuals, and that this toxicity is at its maximum 
after an epileptic seizure. Pellagrini further could demonstrate that 
the cerebrospinal fluid of epileptics is markedly toxic, and that that 
obtained immediately after a convulsion has a toxic and convulsive 
power much greater than that obtained at periods far removed from 
paroxysms. 

Literature. — W. T. Councilman, " Cerebrospinal Meningitis," Johns Hopkins 
Hospital Bull., 1898, p. 27 ; and Phila. Med. Jour., 1898, p. 937. W. T. Councilman, 
F. B. Mallory, and J. H. Wright, "Epidemic Cerebrospinal Meningitis," Am. Jour. 
Med. Sci., 1898, p. 252. W. Osier, " The Cavendish Lecture on the ^Etiology and Diag- 
nosis of Cerebrospinal Fever," Phila. Med. Jour., 1899, p. 26. E. Stadelmann, 
"Meningitis Cerebrospinalis," Zeit. f. kliu. Med., vol. xxxviii. p. 46. B. Neurath, 
Centralbl. f. d. Grenzgebiete d. Med. u. Chir., 1897, vol. i. J. Langer, Jahrb. f. Kin- 
derheilk., 1901, vol. iii. p. 91. Pellagrini, loc. cit., p. 559. 



CHAPTEE X. 
THE EXAMINATION OF CYSTIC CONTENTS. 

CYSTS OF THE OVARIES AND THEIR APPENDAGES. 

The material obtained from cysts of the ovaries or their appen- 
dages varies greatly in character. On the one hand, it may be 
fluid, clear, of low specific gravity, and contain little albumin ; 
while, on the other, it may be dense, viscid, and of colloid appearance. 
The specific gravity varies between 1.018 and 1.024, owing to the 
presence of a large amount of albumin. 

In addition to smaller amounts of serum-albumin and serum- 
globulin the fluid of ovarian cysts contains a considerable quantity 
of another albuminous substance, which is termed metalbumin 
(Scherer) or pseudomucin (Hammarsten). Like Hammarsten's 
mucoid of transudates, it cannot be directly precipitated with acetic 
acid, but must be isolated as follows : The fluid in question is'freed 
from coagulable albumins by boiling after acidifying with acetic acid ; 
the filtrate is precipitated with alcohol, the precipitate dissolved in 
water, dialyzed, and then treated with acetic acid, when the pseudo- 
mucin separates out. The substance contains about 30 per cent, of 
glucosamin. 

Paramucin is another albuminous substance which is found in 
colloid cysts and belongs to the mucinoid bodies. Like the true 
mucins and the body which occurs in exudates the paramucin is 
also precipitated by dilute acetic acid. According to Mitjukoff, it 
contains at least 12.5 per cent, of a reducing substance. 1 

Test for Pseudomucin. — The fluid is mixed with three times its 
volume of alcohol and set aside for twenty-four hours, when it is 
filtered and the precipitate suspended hi water. This is again 
filtered and the filtrate tested in the following manner : 1. A few 
cubic centimeters are boiled, when in the presence of metalbumin 
the liquid will become cloudy, without the formation of a precipitate. 
2. With acetic acid no precipitate is obtained. 3. Upon the appli- 
cation of the acetic acid and potassium ferrocyanide test the liquid 
becomes thick and assumes a yellowish color. 4. When boiled with 

1 Literature dealing with pseudomucin and paramucin : Pseudomucin : Hammarsten, 
Zeit. f. phys. Chem., 1882, vol. vi. p. 194. Pfannenstiel, Arch. f. Gynak., 1890. Zan- 
gerle, Munch, med. Woch., 1900. Paramucin: Mitjukoff, Arch. f. Gynak., 1895. 
Panzer, Zeit. f. phys. Chem., 1899, vol. xxviii. Leathes, Arch. f. exper. Path. u. 
Pharmak., 1899, vol. xliii. 

42 657 



658 



THE EXAMINATION OF CYSTIC CONTENTS. 



Millon's reagent a few cubic centimeters of the nitrate will yield a 
bluish-red color, while the addition of concentrated sulphuric acid, 
without boiling, gives rise to a violet color. 

The color of cystic fluids may vary from a light straw to a reddish 
brown, or even a chocolate ; the latter color may be observed when 
hemorrhage has taken place into the cyst. 

Of morphological elements, ovarian cysts contain red blood-cor- 
puscles, leucocytes, and at times fatty granules in large numbers, 
crystals of cholesterin, hsematoidin, and fatty acids. Most im- 
portant, however, from a diagnostic standpoint is the presence of 
cylindrical or prismatic ciliated epithelial cells, derived from the 
internal lining of the cyst, in the presence of which the diagnosis 
may be definitely made (Fig. 141). At times such cells cannot be 
demonstrated, as they may have undergone fatty degeneration ; 
moreover, if the epithelium lining the cyst is squamous in character, 
it may be difficult, if not impossible, to arrive at a satisfactory con- 

Fig. 141. 




Contents of an ovarian cyst. (Eye-piece III., obj. 8 A, Reichert.) (v. Jaksch.) 
a, Squamous epithelial cells; b, Ciliated epithelial cells; c, Columnar epithelial cells; 
d, Various forms of epithelial cells ; e, Fatty squamous epithelial cells; /, Colloid bodies; 
g, Cholesterin-crystals. 

elusion from an examination of the morphological elements alone. 
Colloid concretions, which may vary in size from several micromil- 
limeters to 0.1 mm., are occasionally observed, and more particu- 
larly in colloid cysts. They may be recognized by their irregular 
form, homogeneous appearance, slightly yellow color, and delicate 
outlines. 

In dermoid cysts, epidermal cells and occasionally hairs are 
observed. 

The differential diagnosis of ovarian, parovarian, and fibrocystic 



HYDRONEPHROSIS. 659 

(uterine) cysts cannot always be made from the character of the fluid 
withdrawn by puncture, but at times it is possible. The most im- 
portant points of difference are here given : 1. The fluid in ovarian 
cystomata is usually more or less viscid, and often contains non- 
nucleated granular corpuscles of about the size of leucocytes, the 
granules of which do not dissolve in acetic acid nor disappear when 
treated with ether. In all probability they are free nuclei ; in the 
United States they are often called Drysdale's corpuscles. 2. In 
parovarian cysts the fluid is thin, watery, of low specific gravity 
(under 1.010), and contains very few morphological elements. 
Cylindrical epithelium is very rarely found during life in the fluid 
withdrawn by aspiration from either ovarian or parovarian cysts. 
3. The fluid from fibrocystic tumors of the uterus is thin, watery, 
and coagulates spontaneously, while that from ovarian and paro- 
varian cysts never coagulates spontaneously unless blood is present. 
Fibrocystic tumors of the uterus have no epithelial lining. 

Of special interest are those cases of ovarian cysts in which in 
the course of typhoid fever infection of the cystic contents occurs 
with the corresponding organism. 1 

HYDATID CYSTS. 

Hydatid cysts are rarely seen in the United States (see page 352). 
The fluid in question is clear, alkaline, of a specific gravity varying 
between 1.006 and 1.010, and contains no albumin. Succinic acid is 
usually present, and may be demonstrated by acidifying a small amount 
of the fluid with hydrochloric acid and evaporating to dryness. The 
residue is extracted with ether and the ether evaporated ; the aqueous 
solution of the second residue, in the presence of succinic acid, will 
yield a rust-colored gelatinous precipitate when treated with a few 
drops of a solution of ferric chloride. Sodium chloride is always 
present in notable amounts, and may be recognized by evaporating 
a drop of the liquid upon a slide, when the characteristic crystals of 
salt will be found. 2 Most important, of course, is the microscopical 
examination, which may reveal the presence of hooklets and shreds 
of membrane, and at times of scolices (see Sputum). 

HYDRONEPHROSIS. 

The diagnosis of hydronephrosis can usually be made without diffi- 
culty if a sufficient amount of fluid can be obtained ; the presence of 
urea and uric acid in notable quantities, as well as of renal epithelial 
cells, which latter especially should be sought for, is quite character- 
istic. Small amounts of uric acid, however, may also be present in 
ovarian cysts. 

1 M. J. Lewis and E. G. Le Conte, Am. Jour. Med. Sci., 1902, vol. cxxiv. p. 590. 

2 J. Munk, Virchow's Archiv, 1875, vol. lxiii. p. 255. 



660 THE EXAMINATION OF CYSTIC CONTENTS. 

PANCREATIC CYSTS. 

These cysts may be recognized by the fact that the fluid possesses 
the power of digesting albumin in alkaline solution. A small 
amount of the liquid is added to milk, when after precipitation of 
the casein the biuret test is applied ; a positive reaction indicates 
the presence of trypsin. Unfortunately, however, the test does not 
always yield positive results, even if the fluid in question is derived 
from a pancreatic cyst, as the trypsin is apparently destroyed in the 
course of time. The larger the cyst, the less likely will it be pos- 
sible to obtain the reaction. A positive result is hence only of 
value, while a negative result does not exclude the existence of the 
disease. 1 

1 Karewski, Deutsch. ined. Woch., 1890, vol. xvi. pp. 1035 and 1069. Hofmeister 
Prag. med. Woch., 1891, vol. xvi. pp. 365 and 377 (see Gussenbauer). v. Jakscn, Zeit. 
f. Heilk., 1888, vol. ix. p. 126 (see Wolfler). 



CHAPTER XI. 

THE SEMEN. 

The ejaculated semen is a mixture of the secretions furnished by 
the testicles, the prostate gland, the seminal vesicles, and the glands 
of Cowper. 

GENERAL CHARACTERISTICS. 

Semen is white or slightly yellowish in color, semifluid, sticky, 
and of an opaque, non-homogeneous, milky appearance, which is due 
to the presence of white, opaque islets floating in the otherwise clear 
fluid ; these consist almost entirely of the specific morphological 
elements of the semen, the spermatozoa. Its odor, which strongly 
resembles that of fresh glue, is characteristic, and is owing to the 
presence of spermin. It is generally attributed to an admixture of 
prostatic fluid, as the semen obtained from the vasa deferentia is 
odorless. According to Robin, however, this odor is produced only 
at the moment of ejaculation, and cannot be ascribed to any single 
one of the secretions present. The reaction of human semen is 
slightly alkaline, and its specific gravity greater than that of water, 
in which it sinks to the bottom. 

CHEMISTRY OF THE SEMEN 

Accurate analyses of human semen or of mammalian semen do 
not exist, and only the old analyses of Vauquelin and Kolliker 
can be given : 



Man. Horse. Ox. 

Water. 90 81.90 82.10 

Albuminous material ~| f ... 15.30 

Extractives .... > . , . . g < 16.45 

Ethereal extract . . j ( ... 2.20 

Mineral material 4 1.61 2.60 



The mineral matter consists largely of calcium phosphate. 

If semen is kept, or if it is slowly evaporated, crystals of phos- 
phate of spermin separate out, which are commonly known as 
Bottcher's crystals, and which were long regarded as identical with 
the so-called Charcot-Leyden crystals that are found in the sputum 
of bronchial asthma, in the blood of leukaemia, in the stools in cases 
of helminthiasis, etc. 

661 



662 



THE SEMEN. 



Spermin is a basic substance, and, according to Ladenburg and 
Abel, is closely related to, if not identical with, diethylene diamin 
(piperazin) : 

° 2H4 \nh/ /C2H4 

The phosphate crystallizes in the form of monoclinic four-sided 
spindles or prisms, which appear as flattened needles of variable 
size. Some are scarcely visible even with a fairly high power of 
the microscope, while others attain the length of 40 p. to 60 //. 
The substance is soluble in formol, thus differing from Charcot- 
Leyden crystals. In water it dissolves with difficulty ; it is slowly 
soluble in acids and alkalies, even in ammonia, while it is insoluble 
in alcohol, ether, chloroform, and dilute saline solution. Florence's 
reagent (see below) colors the crystals a bluish black. According 
to Cohn, the Bottcher crystals are formed exclusively in the prostate 
gland, the gland itself furnishing the basic component, while the 
necessary phosphoric acid is derived from other portions of the 
reproductive apparatus. 1 

MICROSCOPICAL EXAMINATION OF THE SEMEN. 

Upon microscopical examination normal semen is seen to contain 
innumerable actively moving thread-like bodies, measuring from 
50 fj. to 60 [i in length — the spermatozoa. These consist, of an egg- 



Fig. 142. 




Human semen, 
a, Spermatozoa; b, Cylindrical epithelium; c, Bodies enclosing lecithin-granules^ d, 



Squamous epithelium from the urethra ; d', Testicle-cells 
matic crystals ; g, Hyaline globules, (v. Jaksch 



Amyloid corpuscles ; /, Sper- 



shaped head, when seen from above, which is from 3 ji to 5 fi m 
length, the broader end being directed anteriorly ; a middle portion, 
4 fi to 6 fi in length, with which the head is united by its smaller 

1 Th. Cohn, " Zur Kenntniss d. Spermas," Centralbl. f. allg. Path. u. path. Anat., 
vol. x. pp. 940-949. 



THE RECOGNITION OF SEMEN IN STAINS. 663 

end ; and a posterior piece or tail, into which the middle piece grad- 
ually fades (Fig. 142). 

In addition to the spermatozoa a few hyaline bodies are seen which 
are derived from the seminal vesicles ; further, numerous small pale 
granules of an albuminous nature, some testicular and urethral epi- 
thelial cells, lecithin-corpuscles, and so-called prostatic or amyloid 
corpuscles, which at first sight resemble starch-granules in appearance, 
owing to their concentric striations. A few leucocytes and occasion- 
ally a few red corpuscles may also be found. 

PATHOLOGY OF THE SEMEN. 

The study of the semen has received little attention from clini- 
cians, and gynecologists frequently hold the wife responsible for 
sterility when an examination of the husband's semen would — 
according to Kehrer, 1 in 40 per cent. — reveal an absence of sperma- 
tozoa, constituting the condition usually spoken of as azoospermatism. 
This may be temporarily observed following venereal excesses, when 
the fluid finally ejaculated is almost entirely of prostatic origin ; 
their absence then possesses no significance, but persistent azoosper- 
matism must of necessity be associated with sterility. 2 

Cases have been recorded in which, notwithstanding the presence 
of spermatozoa and apparently normal sexual conditions in both 
husband and wife, sterility existed nevertheless, but in which it was 
observed that the spermatozoa lost their motile powder almost imme- 
diately after ejaculation. Under normal conditions, following inter- 
course actively moving spermatozoa may be found in the vagina 
after hours, days, and even weeks. 

Whenever it is deemed advisable to make an examination of the 
semen, this should be done immediately following ejaculation, or as 
soon as possible thereafter. Note should then be taken, not only of 
the presence, but also of the degree of motility of the spermatozoa ; 
a drop of the semen is mixed with a drop of normal (0.6 per cent.) 
saline solution, and examined at once with the microscope. 

Bloody semen, constituting the condition spoken of as hemo- 
spermia, has been observed on several occasions. It may follow 
excessive sexual indulgence, but may also occur in connection with 
gonorrhoeal epididymitis. The blood is readily recognized upon 
microscopical examination. 3 

THE RECOGNITION OF SEMEN IN STAINS. 

In medico-legal cases the physician may be called upon to decide 
whether or not certain stains on body-linen are caused by spermatic 

1 Kehrer, Beitrage z. klin. u. exper. Gynaek., 3879, vol. ii., Giessen. 

2 Furbringer, Zeit. f. klin. Med., 1881, vol. iii. p. 310. 

3 Feleki, Centralbl. f. Krankh. d. Hani- u. Sexualorgane, 1901, vol. xii. p. 503. 



664 THE SEMEN. 

fluid, whether or not a rape has been committed, etc. In such cases 
it is frequently only necessary to examine a drop of the vaginal 
fluid in order to arrive at a positive result at once. At other times, 
however, recourse must be had to the following method : a fragment 
of the linen or scrapings from the vulva or vagina are placed in a 
watch-crystal and allowed to soak for at least one hour in from 27 
to 30 per cent, alcohol, when a bit of the material is teased in a 
solution of eosin in glycerin (1 : 200), and examined. The 
heads of the spermatozoa are thus stained a deep red, while the 
tails, which are often broken, exhibit a pale-rose tint, and can 
readily be distinguished from vegetable fibres, which do not take 
the stain at all. A positive statement can thus be made in every 
case, even after months and years, as spermatozoa not only resist the 
action of reagents, but also the process of putrefaction ; this is prob- 
ably owing to the large proportion of mineral matter which enters 
into their composition, and which insures preservation of their form. 
Instances have been recorded in which it was possible to demon- 
strate spermatozoa in stains after eighteen years. 

The semen test of Florence 1 has attracted much attention, and 
may be recommended in doubtful cases ; only a negative result, 
however, is of value (see below). It is based upon the observation 
that very characteristic crystals of iodospermin are formed when 
spermatic fluid is treated with a solution of iodo-potassic iodide 
containing 1.65 grammes of pure iodine and 2.54 grammes of 
potassium iodide, dissolved in 26 c.c. of water. When a drop 
of this solution is added to a drop of spermatic fluid or an aqueous 
extract of a seminal stain, dark-brown crystals of iodospermin sepa- 
rate out at once, and may be readily recognized under the microscope. 
They occur in the form of long rhombic platelets or fine needles, 
often grouped in rosettes, but also occurring singly or as twin 
crystals. The examination with the microscope should be made at 
once after addition of the reagent, as the crystals disappear on 
standing. 

As the reaction may also be obtained in cases of azoosperma- 
tism, and with pure prostatic secretion, while a negative result is 
obtained with the fluid from spermatoceles, it is manifest that the 
test is not applicable for the determination of the presence or ab- 
sence of spermatozoa per se. Posner 2 states that he obtained 
similar crystals when the test was applied to a glycerin extract of 
ovaries. 

More recently Eichter 3 has shown that Florence's reaction is also 
obtained with a decomposition-product of lecithin, viz., choliu, 

1 Florence, " Du sperme et des taches de sperme en medecine legale," Arch. 
d'Anthrop. crimin., vols. x. and xi. 

2 C. Posner, " Die Florence' sche Keaktion," Berlin, klin. Woch., 1897, p. 602. 

3 M. Eichter, " D. mikrochemische Nachweis v. Sperma," Wien. klin. Woch., 1897, 
p. 569. 



THE RECOGNITION OF SEMEN IN STAINS. 665 

which would explain the observation that better results are com- 
monly obtained with dried semen than with fresh material. But 
it follows also that the reaction cannot be a specific semen reaction, 
and Richter accordingly concludes that a negative result only is of 
value, and indicates that the material under examination is not 
semen. He states that he obtained positive results with vaginal 
and uterine mucus, with decomposing brain-substance, and other 
organs as well. In confirmation of Richter's results, Bocarius ! has 
demonstrated that the so-called iodospermin is in reality an iodized 
product of cholin and not of spermin. 

1 N. Bocarius, Zeit. f. phys. Cheni., 1902, vol. xxxiv. p. 339. 



CHAPTEE XII. 
VAGINAL DISCHARGES. 

GENERAL CHARACTERISTICS. 

The secretion which is normally furnished by the vaginal glands 
is small in amount, and just sufficient to keep the mucous mem- 
brane moist. It is a clear or somewhat milky -looking, semiliquid 
material, in which numerous epithelial laminae, which have been 
thrown off during the normal process of desquamation, may be 
found. It has been stated that the reaction of the vaginal secretion 
in virgins is invariably acid, while an alkaline reaction is the rule in 
the d&jlorees. During pregnancy, however, the secretion is probably 
always acid. In five hundred cases which Kronig examined in this 
direction an alkaline reaction was never observed. According to 
Zweifel, the vaginal secretion contains traces of trimethylamin. 1 

Microscopically, immerous epithelial cells, mucous corpuscles, a 
few large mononuclear leucocytes cellular detritus, and bacteria are 
found (Fig. 143). Doderlein 2 has described a non-pathogenic 

Fig. 143. 




Vaginal secretion, 
a, Mucous corpuscles ; b, Vaginal epithelium ; c, Epithelium from vulva. 



bacillus or a group of bacilli which are characterized by the fact 
that they give rise to marked acid fermentation of sugar, and he 
regards these organisms as the only ones which are constantly 
present in the normal vagina. Kronig and Menge, however, state 



1 Zweifel, Arch. f. Gynaek., 1881, vol. xviii. p. 359. 

2 Doderlein, Ibid., 1887, vol. xxxi. p. 412. 



666 



GENERAL CHARACTERISTICS. 667 

that they are often absent. These observers have found, on the 
other hand, that under normal conditions there are various bacilli and 
cocci present which belong to the class of obligatory anaerobes, and 
are likewise non-pathogenic. Unfortunately they have not described 
these organisms in detail. Near the outlet they found bacteria which 
may be cultivated upon alkaline aerobic culture-media, but which are 
usually absent in the upper portion of the vagina. 

It is important to note that various diplococci may also be found 
under normal conditions, and care should be taken not to confound 
these with gonococci. Like the gonococci, they are decolorized by 
Gram's method. If the characteristics of the former be borne in 
mind, however, mistakes may probably always be avoided ; in mar- 
ried women and in children it is best to make the diagnosis of gon- 
orrhoea only when the gonococcus has been isolated by cultivation. 

The question whether or not pathogenic bacteria may occur in the 
normal vagina of pregnant or non-pregnant Avomen, may be an- 
swered in the affirmative ; but with the exception of the gonococcus 
they are not often seen. 1 Bergholm 2 thus recently examined 
the vaginal secretion of forty pregnant women, and was unable 
to obtain organisms pathogenic for animals in a single case. There 
were no pyogenic staphylococci, no streptococci, and no colon bacilli. 

The vaginal secretion has been shown to possess powerful bac- 
tericidal properties, so that pathogenic organisms, even when arti- 
ficially introduced into the vagina, are rapidly killed. Kronig thus 
found that the bacillus pyocyaneus disappears from the vagina of 
pregnant women in from ten to thirty hours, the staphylococci in 
from six to thirty-six hours, and the streptococcus pyogenes within 
six hours. Important from a practical standpoint is the fact that 
the bacteria disappeared less rapidly when irrigation of the vagina 
with water or even antiseptics was employed. 

Of animal parasites, the Trichomonas vaginalis is occasionally 
encountered in the vaginal discharge. The organism is identical 
with the trichomonas found in the feces and the urine. In the 
United States it is not so common as among the peasant population 
of Central Europe. As far as is known, the organism is of no patho- 
logical significance. From a medicolegal standpoint, however, its 
presence may not be unimportant, as cases are on record in which 
trichomonades have been confounded with spermatozoa. In my 
judgment, however, such a mistake can only occur if the observer 
is without training in microscopy. 

The possible presence of the anguillula aceti in the vaginal dis- 
charge has been pointed out by Billings, Miller, and Stiles. Stiles 
has suggested that it may be introduced into the vagina by injections 
of vinegar water taken with the object of preventing conception. 

1 Doderlein, Das Scheidensecret, Leipzig, 1892. J. W. Williams, Am. Jour. Obstet., 
1898, vol. xxxviii. ; Trans. Am. Gyn. Soc, 1898; Am. Jour. Obstet., 1898. 

2 H. Bergholm, Arch. f. Gynak., 1902, vol. lxvi. Heft 3. 



668 VAGINAL DISCHARGES. 

VAGINAL BLENNORRHEA. 

In physiological conditions an increased vaginal secretion is ob- 
served during sexual excitement, especially during coitus, just pre- 
ceding and at the beginning of menstruation, and during preg- 
nancy, when a profuse blennorrhea is frequently seen, which often 
assumes a virulent character. The secretion under such conditions 
readily becomes purulent. When not dependent upon a gonorrheal 
infection the secretion is thicker than normal, white, and creamy. 
At times also the vaginal catarrh observed in pregnancy is com- 
plicated with mycosis, when white or yellowish-gray patches may 
be seen at the orifice of the vagina ; the latter may, indeed, even 
be filled with particles which consist entirely of fungi. 

MENSTRUATION. 

At the beginning of menstruation, as has been pointed out above, 
an increase in the amount of vaginal secretion is observed, in which 
leucocytes, prismatic epithelial cells coming from the uterus, as well 
as the usual vaginal cells, may be seen upon microscopical exami- 
nation. Later the secretion becomes sanguineous in character, 
and finally only epithelial cells, leucocytes, and granular detritus are 
encountered, the cells usually showing evidence of fatty degenera- 
tion. The amount of blood lost at each menstrual period amounts 
to about 200 grammes in perfectly healthy females. 

THE LOCHIA. 

The lochia during the first day following parturition are red in 
color — the lochia rubra — and emit the characteristic sanguineous 
odor. At this time a microscopical examination will reveal an 
abundance of red corpuscles, some leucocytes, and a variable number 
of epithelial cells, which are almost exclusively of vaginal origin. 
On the second and third days the number of red corpuscles dimin- 
ishes, while the leucocytes increase in number. Still later the dimi- 
nution in the red and the increase in the white corpuscles become 
more marked, and the discharge at the same time assumes a grayish 
or white color, until about the tenth day the red corpuscles have 
almost entirely disappeared, while the leucocytes and epithelial cells 
are abundant. Finally, the secretion becomes thicker, mucoid, and 
milky white in color — the lochia alba, which condition may persist 
for from three to four weeks in nursing-women, and still longer in 
those who do not nurse, until finally the normal secretion is again 
established. Numerous bacteria are encountered in the lochia, and 
it is curious to note that among these pus-organisms are quite con- 
stantly present without giving rise to symptoms. When a portion 



VULVITIS AND VAGINITIS— GONORRHCEA. 669 

of the placenta or membranes have been retained the lochia soon 
give off a fetid odor, and assume a dirty brownish color ; the reten- 
tion of blood-clots alone may also produce this result. In such 
cases the lochia swarm with bacteria of all kinds. 1 

VULVITIS AND VAGINITIS. 

In cases of vulvitis and vaginitis a marked increase is observed 
in the number of the leucocytes and epithelial cells, the character of 
the latter depending, essentially of course, upon the portion of the 
genital tract affected. Red corpuscles are also met with at times ; 
their number generally stands in a direct relation to the intensity of 
the inflammatory process. In some instances epithelial casts of 
the entire vagina have been observed, constituting the condition 
termed vaginitis exfoliativa. The condition, however, is rare. 

The discharge of large amounts of pure pus through the vagina 
points to perforation of an abscess of the genital organs or of the 
neighboring structures into the uterus or the vagina ; it is of rare 
occurrence. Much more common is the discharge of fecal matter 
or of urine through this channel, indicating the existence of a 
vagino-rectal or vagino-vesical fistula. 

MEMBRANOUS DYSMENORRHEA. 

While ordinarily, during menstruation, shreds of desquamated 
uterine lining are frequently encountered, it is rare to meet with 
large pieces or complete casts of the uterus, the elimination of which 
is usually associated with the symptoms of a severe dysmenorrhea, 
constituting the condition spoken of as membranous dysmenorrhea. 

CANCER. 

While the diagnosis of malignant growth of the uterus is probably 
never based upon a microscopical examination of the vaginal dis- 
charge alone, it may be mentioned that in advanced cases this is pos- 
sible, as fragments of an epithelioma of the cervix, for example, may 
frequently be detected upon microscopical examination (Fig. 144). 
In suspected cases small pieces of tissue should be removed and 
examined according to usual histological methods. 2 

GONORRH(EA. 

In suspected cases of gonorrhoea an examination of the vaginal 
and urethral discharge for the presence of gonococci is important, 
as it is practically impossible to diagnose this condition posi- 
tively in any other manner. Care should be taken, however, not to 

1 Doderlein, loc. cit. Thomen, Centralbl. f. d. med. Wiss., 1890, vol. xxviii. p. 537; 
and Arch. f. Gyn., 1889, vol. xxxvi. p. 231. 

2 T. S. Cullen, Cancer of the Uterus, Appleton & Co., 1900. 



670 



VAGINAL DISCHARGES. 
Fig. 144. 




Vaginal secretion from a case of epithelioma of the cervix uteri. 

confound the diplococci which may be normally present in the 
urethra and vagina with gonococci (see chapter on the Urine). 

ABORTION. 

In cases of abortion it is often possible to discover chorion villi in 
the expelled blood-clots which present the characteristic capillary 

Fig. 145. 




Chorion villi. 



ABORTION. 671 

network (Fig. 145), and often manifest signs of advanced fatty 
degeneration. Important also from a diagnostic point of view is the 

Fig. 146. 




Decidual cells. 



presence of decidual cells (Fig. 146), which are characterized by 
their large size, their round, polygonal, or spindle-like form, and 
their characteristic nuclei and nucleoli. 



CHAPTEE XIII. 
THE SECRETION OF THE MAMMARY GLANDS. 

THE SECRETION OF MILK IN THE NEWLY BORN. 

A secretion from the mammary glands of the male is observed 
only in the newly born, if we except those rare cases in which 
adult males were known to suckle infants. The fluid in question, 
which may also be obtained from the female infant, is termed 
" Hexenmilch " (witches' milk) by the Germans. Qualitatively it 
has the same composition as milk, but may manifest considerable 
quantitative variations. 

COLOSTRUM. 

Aside from those curious instances in which a secretion of milk 
has been observed in non-pregnant women, mammary activity is 
essentially connected with the physiological phenomena of pregnancy 
and parturition. Often as early as the third month a small drop of 
a serous-looking fluid can be obtained from the nipple by pressure 
upon the breasts. Immediately after delivery a variable amount of 
fluid is secreted, which is watery, semi-opaque, mucilaginous, and 
of a yellowish color. To this secretion, as well as to that observed 
during pregnancy, the term colostrum has been applied. It is dis- 
tinguished from true milk by its physical characteristics and by the 
presence of a greater proportion of sugar and salts. The fluid, 
moreover, coagulates upon boiling. An idea may be formed of its 
composition from the appended table : 





4 weeks before birth. 


17 days be- 


9 days be- 


24 hours 


2 days 




I. 


ii. 


fore birth. 


fore birth. 


after birth. 


after birth. 


Water . . . 


945.2 


852.0 


851.7 


858.8 


843.0 


867.9 


Solids . . . 


54.8 


148.0 


148.3 


141.2 


157.0 


132.1 


Casein . . . 












21.8 


Albumin . . 


28.8 


69.0 


74.8 


80.7 






Fat .... 


7.3 


41.3 


30.2 


23.5 




48.6 


Lactose . . . 


17.3 


39.5 


43.7 


36.4 




61.0 


Salts .... 


4.4 


4.4 


4.5 


5.4 


5.1 




Upon mk 


;roscopica 


I examina 


Aon fat-( 


Iroplets, 


a few le 


ucocytes, 



some epithelial cells, and so-called colostrum-corpuscles are found. 

672 



HUMAN MILK. 
Fig. 147. 





673 




Colostrum of a woman in sixth month of pregnancy. (Eye-piece III., obj. 8 A., Reichert.) 

(v. Jaksch.) 

The latter are highly refractive bodies, of irregular size, whose inte- 
rior is filled with fatty granules (Fig. 147). 

Literature.— G. Woodward, Jour. Exper. Med., vol. ii. p. 217. 

THE SECRETION OF MILK PROPER, IN THE ADULT 

FEMALE. 

The secretion of milk proper usually begins about the third day 
following parturition, and may continue for a variable length of 
time. On the one hand, the amount of milk secreted may be so 
small as to be insufficient for the needs of the child, so that lacta- 
tion may have to cease after several days; on the other hand, 
women are not infrequently seen who nurse their children for two 
years and even longer. Usually infants are nursed until six or seven 
teeth have appeared, which period varies with the individual child, 
averaging about the eleventh month. 

HUMAN MILK. 

Human milk is of a bluish color, and differs in this respect from 
the milk of cows. Its reaction is alkaline. The specific gravity 
may vary between 1.026 and 1.035, one between 1.028 and 1.034 
being the most common. The amount of milk secreted in twenty- 
four hours varies from 500 to 1500 c.c. Microscopically, it is a 
fairly homogeneous emulsion of fat, and is practically destitute of 
cellular elements. From the following table an idea may be formed 
of its chemical composition : 





Biehl. 


Gerber. . 


Christenn. 


Pfeiffer. 


Pfeiffer. 


Mendes de 
Leon 


Water ...... 

Solids ..... 

Albumin .... 

Fat 

Lactose 

Salts 


876.00 

124.00 

22.10 

38.10 

60.90 

2.90 


891.00 

109.00 

17.90 

33.00 

53.90 

4.20 


872.40 

127.60 

19.00 

43.20 

59.80 

2.60 


892.00 

108.00 

16.13 

32.28 

57.94 

1.65 


890.60 

109.40 

17.24 

29.15 

59.92 
2.09 


877.90 

'25.30 

38.90 

55.40 

2.50 



Upon comparing this table with the following analysis of cows 7 
milk it will be seen that the latter contains more albumin and less sugar 
43 



674 THE SECRETION OF THE MAMMARY GLANDS. 

than human milk. Human milk, moreover, is relatively deficient in 
mineral matter, and especially in calcium salts and phosphoric acid : 

Water 874.2 

Solids 125.8 

Casein 28.8) QA K 

Albumin 5.3/ 34 ' 5 

Fat 36.6 

Lactose 48. 1 

Salts 7.1 

Of inorganic salts human milk contains about 0.7 pro mille of 
potassium (K 2 0), 0.2 of sodium, 0.3 of calcium, 0.06 of magnesium, 
from 3.52 to 7.21 mgrms. of iron, about 0.4 pro mille of phosphoric 
acid and 0,4 of chlorine. 

The albumins found in milk-plasma are casein, lactoglobulin, 
and lactalbumin. It is claimed by some observers that the casein 
of human milk differs from that obtained from cows' milk. The 
casein-coagula in human milk are not so large and dense as those 
observed in cows' milk. Human casein, moreover, is not so readily 
precipitated by acids and salts ; it does not always coagulate upon 
the addition of rennet ferment, and while it may be precipitated by 
the gastric juice, it is readily dissolved by an excess. Although 
accurate analyses of human casein are not available, it is probable 
that the two forms are not identical (Hammarsten). 

The question whether or not normal human milk contains micro- 
organisms may now be answered in the affirmative. There can be 
no doubt, however, that the milk as it is secreted by the healthy 
gland is sterile, but upon passing along the lacteal ducts in the 
nipple it is always contaminated by the Staphylococcus epidermidis 
albus (Welch). This micro-organism must be regarded as a constant 
inhabitant of the skin, and is the only one of the cutaneous bacteria 
which penetrates the deeper layers of the epidermis and the gland- 
ular appendages of the skin. It is thus apparent Avhy this organism 
is so constantly met with, and is practically the only one found in 
normal human milk. Exceptionally the Staphylococcus pyogenes 
aureus is found. 

THE MILK IN DISEASE. 

The chemistry of the milk in pathological conditions has received 
little attention. It appears, however, that the milk of women when 
ill usually contains less fat, and that the proportion of lactose is 
diminished. In cases of jaundice the presence of bile-pigment and 
of biliary acids has not been satisfactorily demonstrated. According to 
Friedjung, 1 a subnormal amount of iron is usually found in the milk 
when nurslings do not thrive on apparently normal milk. In cases 
of mammary tumors bloody secretion has been observed in rare 
cases, the nipple itself being intact. 

Microscopically, an admixture of leucocytes is observed in various 

1 G. K. Friedjung, Arch. f. Kinderheilk., vol. xxxi. Hefte 1 u. 2. 



. 



THE MILK IN DISEASE. 



675 



Ftg. 148. 



TQ 



diseases of the breast, and especially in cases of abscess. Of patho- 
genic micro-organisms, streptococci may be found in cases of puer- 
peral fever ; more commonly, however, they are absent. The 
typhoid bacillus has occasionally been seen in 
cases of typhoid fever, and it is interesting to 
note that the specific agglutinins of typhoid fever 
have been found in the milk. Pueumococci have 
been obtained from the milk of pregnant women 
affected with lobar pneumonia. The important 
question whether or not tubercle bacilli are elimi- 
nated in the milk in cases of phthisis cannot be 
definitely answered. In cows such an occurrence 
is certainly common, even when there is no demon- 
strable tubercular lesion of the udder. So far as 
I have been able to ascertain, however, tubercle 
bacilli have never been found in human milk. 1 

A blue and a red color have been observed 
in the milk of cows, owing to the presence of the 
Bacillus pyocyaneus and the Micrococcus prodig- 
iosus, respectively. 

A chemical examination of human milk should 
always be made whenever it is apparent that 
the nutrition of the baby is below normal. 
Valuable dietetic suggestions may thus be ob- 
tained. In other cases, as when the mother is 
unwilling or unable to nurse her child beyond a 
certain period, a knowledge of the composition of 
her milk will enable the physician to give specific 
instructions regarding the proper modification of 
cows' milk. If a wet-nurse is to be employed, 
her milk should likewise be examined. 

Most important is the determination of the 
specific gravity and of the amount of fat. The 
former may vary between 1.029 and 1.033. The 
amount of fat should not be less than 3 per cent. 

Determination of the Specific Gravity. 

The sp. gr. is best determined with the lacto- 
densimeter of Quevenne (Fig. 148). As the in- 
strument is graduated for a temperature of 60° F., 
it is necessary to correct the sp. gr. whenever the 
temperature is above or below this point. In 
the following tables the corrected sp. gr. may be 
found corresponding to temperatures ranging from 46° to 75° F. : 

1 Escherich, Fortscbr. d. Med., 1885, vol. iii. p. 321. Karlinski, Wien. nied. Woch., 
1888, vol. xxxviii. No. 28. Ott, Prag. med. Woch.. 1892, vol. xvii. p. 145. Cohn u. 
Neumann, Virchow's Archiv, 1880, vol. cxxvi. p. 187. 




Quevenne's lactoden- 
simeter. 



676 THE SECRETION OF THE MAMMARY GLANDS. 

Corrections for Temperature. 



Specific 






Degrees of thermometer (Fahrenheit). 






gravity. 


46 


47 


48 


49 


50 


51 


52 


53 


54 


55 


1020 


19.0 


19.1 


19.1 


19.2 


19.2 


19.3 


19.4 


19.4 


19.5 


19.6 


1021 


20.0 


20.0 


20.1 


20.2 


20.2 


20.3 


20.3 


20.4 


20.5 


20.6 


1022 


21.0 


21.0 


21.1 


21.2 


21.2 


21.3 


21.3 


21.4 


21.5 


21.6 


1023 


22.0 


22.0 


22.1 


22.2 


22.2 


22.3 


22.3 


22.4 


22.5 


22.6 


1024 


22.9 


23.0 


23.1 


23.2 


23.2 


23.3 


23.3 


23.4 


23.5 


23.6 


1025 


23.9 


24.0 


24.0 


24.1 


24.1 


24.2 


24.3 


24.4 


24.5 


24.6 


1026 


24.9 


24.9 


25.0 


25.1 


25.1 


25.2 


25.2 


25.3 


25.4 


25.5 


1027 


25.9 


25.9 


26.0 


26.1 


26.1 


26.2 


26.2 


26.3 


26.4 


26.5 


1028 


26.8 


26.8 


26.9 


27.0 


27.0 


27.1 


27.2 


27.3 


27.4 


27.5 


1029 


27.8 


27.8 


27.9 


28.0 


28.0 


28.1 


28.2 


28.3 


28.4 


28.5 


1030 


28.7 


28.7 " 


28.8 


28.9 


29.0 


29.1 


29.1 


29.2 


29.4 


29.4 


1031 


29.6 


29.6 


29.7 


29.8 


29.9 


30.0 


30.1 


30.2 


30.3 


30.4 


1032 


30.5 


30.5 


30.6 


30.7 


30.9 


31.0 


31.1 


31.2 


31.3 


31.4 


1033 


31.4 


31.4 


31.5 


31.6 


31.8 


31.9 


32.0 


32.1 


32.3 


32.4 


1034 


32.3 


32.3 


32.4 


32.5 


32.7 


32 9 


33.0 


33.1 


33.2 


33.3 


1035 


33.1 


33.2 


33.4 


33.5 


33.6 


33.8 


33.9 


34.0 


34.2 


34.3 



Specific 


Degrees of thermometer (Fahrenheit). 


gravity. 


56 


57 


58 


59 


60 


61 


62 


63 


64 


65 


1020 


19.7 


19.8 


19.9 


19.9 


20.0 


20.1 


20.2 


20.2 


20.3 


20.4 


1021 


20.7 


20.8 


20.9 


20.9 


21.0 


21.1 


21.2 


21.3 


21.4 


21.5 


1022 


21.7 


21.8 


21.9 


21.9 


22.0 


22.1 


22.2 


22.3 


22.4 


22.5 


1023 


22.7 


22.8 


22.8 


22.9 


23.0 


23.1 


23.2 


23.3 


23.4 


23.5 


1024 


23.6 


23.7 


23.8 


23.9 


24.0 


24.1 


24.2 


24.3 


24.4 


24.5 


1025 


24.6 


24.7 


24.8 


24.9 


25.0 


25.1 


25.2 


25.3 


25.4 


25.5 


1026 


25.6 


25.7 


25.8 


25.9 


26.0 


26.1 


26.2 


26.3 


26.5 


26.6 


1027 


26.6 


26.7 


26.8 


26.9 


27.0 


27.1 


27.3 


27.4 


27.5 


27.6 


1028 


27.6 


27.7 


27.8 


27.9 


28.0 


28.1 


28.3 


28.4 


28.5 


28.6 


1029 


28.6 


28.7 


28.8 


28.9 


29.0 


29.1 


29.3 


29.4 


29.5 


29.6 


1030 


29.6 


29.7 


29.8 


29.9 


30.0 


30.1 


30.3 


30.4 


30.5 


30.7 


1031 


30.5 


30.6 


30.8 


30.9 


31.0 


31.2 


31.3 


31.4 


31.5 


31.7 


1032 


31.5 


31.6 


31.7 


31.9 


32.0 


32.2 


32.3 


32.5 


32.6 


32.7 


1033 


32.5 


32.6 


32.7 


32.9 


33.0 


33.2 


33.3 


33.5 


33.6 


33.8 


1034 


33.5 


33.6 


33.7 


33.9 


34.0 


34.2 


34.3 


34.5 


34.6 


34.8 


1035 


34.5 


34.6 


34.7 


34.9 


35.0 


35.2 


35.3 


35.5 


35.6 


35.8 



Specific 


Degrees of thermometer (Fahrenheit). 


gravity. 


66 


67 


68 


69 


70 


71 


72 


73 


74 


75 


1020 


20.5 


20.6 


20.7 


20.0 


21.0 


21.1 


21.2 


21.3 


21.5 


21.6 


1021 


21.6 


21.7 


21.8 


22.0 


22.1 


22.2 


22.3 


22.4 


22.5 


22.6 


1022 


22.6 


22.7 


22.8 


23.0 


23.1 


23.2 


23.3 


23.4 


23.5 


23.7 


1023 


23.6 


23.7 


23.8 


24.0 


24.1 


24.2 


24.3 


24.4 


24.6 


24.7 


1024 


24.6 


24.7 


24.9 


25.0 


25.1 


25.2 


25.3 


25.5 


25.6 


25.7 


1025 


25.6 


25.7 


25.9 


26.0 


26.1 


26.2 


26.4 


26.5 


26.6 


26.8 


1026 


26.7 


26.8 


27.0 


27.1 


27.2 


27.3 


27.4 


27.5 


27.7 


27.8 


1027 


27.7 


27.8 


28.0 


28.1 


28.2 


28.3 


28.4 


28.6 


28.7 


28.9 


1028 


28.7 


28.8 


29.0 


29.1 


29.2 


29.4 


29.5 


29.7 


29.8 


29.9 


1029 


29.8 


29.9 


30.1 


30.2 


30.3 


30.4 


30.5 


30.7 


30.9 


31.0 


1030 


30.8 


30.9 


31.1 


31.2 


31.3 


31.5 


31.6 


31.8 


31.9 


32.1 


1031 


31.8 


32.0 


32.2 


32.2 


32.4 


32.5 


32.6 


32.8 


33.0 


33.1 


1032 


32.9 


33.0 


33.2 


33.3 


33.4 


33.6 


33.7 


33.9 


34.0 


34.2 


1033 


33.9 


34.0 


34.2 


34.3 


34.5 


34.6 


34.7 


34.9 


35.1 


35.3 


1034 


34.9 


35.0 


35.2 


35.3 


35.5 


35.6 


35.8 


36.0 


36.1 


36.3 


1035 


35.9 


36.1 


36.2 


36.4 


36.5 


36.7 


36.8 


37.0 


37.2 


37.3 



THE MILK IN DISEASE. 



Estimation of the Fat. 



The estimation of the fat is most conveniently made by means of 
the lactoscope of Feser, shown in Fig. 149. Milk is drawn into 

Fig. 149. 




Feser's lactoscope. 



the pipette up to the mark M, when it is emptied into the cylinder 
C. The pipette is then rinsed with water and the washings added 
to the milk. While shaking, water is added until the black lines 
upon the milk-colored glass plug J. can just be discerned. The fig- 
ure upon the right of the scale at the level reached by the mixture 
indicates the percentage-amount of fat, while the number upon the 
left indicates in cubic centimeters the amount of water that has 
been added. 

Estimation of the Proteids. 

Woodward's Method. — Two " milk-burettes " (see Fig. 150), 
each containing 5 c.c. of milk, are kept at a temperature of from 37° 
to 40° C. for from eighteen to twentv-four hours. At the end of 



678 



THE SECRETION OF THE MAMMARY GLANDS. 



this time the milk has separated into two layers, viz., an upper layer 
of viscid yellow fat, and a lower layer of fluid milk, which is quite 
opaque above and almost translucent below. Clinging to the sides 
of the tube, and especially at the bottom, a granular precipitate will 
be seen. The burettes are then cooled, when the milk-serum is 



Fig. 150. 




Woodward's milk-burette. 

withdrawn into two tubes graduated to 15 c.c, and treated with 
Esbach's reagent to the 15 c.c. mark. The mixture in each tube is 
thoroughly stirred with a glass rod and then centrifugated to a con- 
stant reading. 

Woodward l has checked his analyses by Kjeldahl's method, and 
has obtained satisfactory results. 

1 G. Woodward, " A Clinical Method for the Estimation of Breast-milk Proteids," 
Phila. Med. Jour., 1898, p. 956. 



INDEX 



ABOBTION, vaginal discharge in, 670 
Absorption, rate of, in the stomach, 
268 
Acetic acid, 255 

fermentation, 254 
tests for, 255 
Acetonemia, 54 
Acetone in the blood, 54 

in the gastric contents, 258 
in the urine, 568 
quantitative estimation of, 571 
tests for, 569 
Acetonuria, 568 
Acholic stools, 287, 295 
Achroodextrin, 199, 245 
Acid, acetic, 255, 281 
benzoic, 457 
butyric, 254, 281 
carbolic, 279, 566 
diacetic, 573 

diazo-benzene-sulphonic, 559 
formic, 281 
glucuronic, 533 
hippuric, 457 
homogentisinic, 555 
hydrochloric, 217 
lactic, 245 
oxalic, 466 
oxaluric, 466 
oxybutyric, 574 
phosphoric, 395 
propionic, 281 
succinic, 659 
sulphuric, 404 
tauro-carbaminic, 411 
uric, 442 
uroleucinic, 555 
valerianic, 282 
Acids, organic, in the gastric contents, 

253 
Acquisto's fluid, 142 
Actinomyces hominis, 203, 362, 627 
Actinomycosis, 203, 362 
Adenin in the urine, 455 
Afanassiew's reagent, 113 
Agglutinins, 170 
a-granulation of Ehrlich, 79 
Albumin, aceto-soluble, 485 
in the feces, 333 
in the urine, 472 
quantitative estimation of, 499 
special test for serum-albumin, 498 
for serum-globulin, 502 



Albumin, tests for, 492 
boiling, 496 
nitric acid, 493 
picric acid, 498 
potassium ferrocyanide, 497 
Spiegler's, 498 
trichloracetic acid, 497 
Albuminimeter, 500 
Albuminous expectoration, 366 
Albuminuria, 472 
accidental, 484 
colliquative, 479 
constitutional, 475 
cyclic, 474 
Da Costa's, 476 
digestive, 483 
febrile, 477 
functional, 474, 476 
hematogenous, 475, 481 
in organic diseases of the kidneys, 

477, 484 
intermittent, 474 
mixed, 484 
neurotic, 482 
orthostatic, 474 
physiological, 472 
postural, 474 

referable to circulatorv disturbances, 
480 
to impeded outflow of urine, 481 
renal, 477, 484 
toxic, 482 
transitory, 474 
Albumoses in the blood, 44 
in the feces, 333 
in the gastric contents, 244 
in the urine, 486 
tests for, 502 
Albumosuria, 486 
digestive, 487 
enterogenic, 486 
hematogenic, 486 
hepatogenic, 486 
histogenic, 486 
mixed, 487 
pyogenic, 486 
renal, 486 
vesical, 486 
Alkalimeter, Engel's, 25 
Alkaline stools, 278, 286 

urine, 381 
Alkalinity of the blood, 20 
distribution of, 139 

679 



680 



INDEX 



Alkalinity of the blood, estimation of, 

21 
Alkapton in the urine, 555 
Alkaptonuria, 555 
Alloxur bases in the urine, 442, 455 

estimation of, 456 
Almen's solution, 518 
Alveolar epithelium, 349 
Ammonia in the blood, 47 

in the gastric contents, 257 

in the urine, 439 

estimation of, 441 
Ammoniacal fermentation, 382 
Ammonio-magnesium phosphate, 591 
Ammonium urate, 601 
Amoeba coli, 299 

in the feces, 299 
in the sputum, 353 
Amoebae in the urine, 629 
Amoebinae in the feces, 298 
Amphistomum hominis, 315 
Amphoteric urine, 381 
Amyloid corpuscles in the semen, 663 
Anachlorhydria, 224 
Anacidity, hysterical, 224 
Anadeny of the stomach, 224 
Anaemic degeneration of the red corpus- 
cles, 63 
Ankylostomiasis, 317 
Anchylostomum duodenale, 317 
Anguillula aceti, 629, 667 

intestinalis, 320, 321 

stercoralis, 320, 321 
Anguilluliasis, 197, 320 
Anilin dyes, classification of, 119 

water, gentian-violet, 207 
Animal gum in the urine, 533 

parasites in the blood, 182 
in the feces, 297 
in the sputum, 352 
in the urine, 628 
Annelides, 315 

Anthracosis of the lungs, 365 
Anthrax, bacillus of, 177 
Arabinose in urine, 532 
Arnold's test for diacetic acid, 573 
Ascarides in the feces, 315 

in the urine, 629 
Ascaris lumbricoides, 315 

maritima, 316 

mystax, 316 
Asiatic cholera, bacillus of, 323 
Asthma, bronchial, Charcot-Leyden crys- 
tals in, 364 
Azoospermatism, 663 

BACILLI of Booker, 325 
Bacillus acidophilus, 329 
coli communis, 328 
dysenteriae, 331 
lactis aerogenes 328 
melitensis, 180 
of anthrax, 177 



Bacillus of cholera Asiatica, 323 
of diphtheria, 206 
of dysentery, 331 
of Finkler and Prior, 325 
of glanders, 178 
of influenza, 178, 360 
of leprosy, 356 
of Le Sage, 325 
of Malta fever, 180 
of Moro, 329 
of Oppler and Boas, 264 
of paratyphoid fever, 170 
of plague, 181, 362 
of Shiga, 331 

of tuberculosis, in the blood, 177 
in the feces, 328 
in the gastric contents, 264 
in the meningeal fluid, 654 
in the milk, 675 
in the mouth, 203 
in the nasal discharge, 336 
in the sputum, 354 
in the urine, 626 
methods of staining, 357 
of typhoid fever, in the blood, 165 
in the feces, 325 
in the sputum, 362 
in the urine, 625 
of whooping-cough, 361 
of yellow fever, 180 
pestis, 181, 362 
pyocyaneus, 329 
smegma, 356, 361 
Bacteria in blood, 165 
in exudates, 635 
in feces, 276, 323 
in gastric contents, 264 
in milk, 675 
in mouth, 203 
in nasal secretion, 336 
in pus, 642 
in sputum, 354 
in urine, 626 
in vagina, 667 
Bacterial decomposition of the urine, 382 
Bacteriuria, 623 

idiopathic, 627 
Bacterium lactis aerogenes, 328 
Balantidium coli, 304 
Bang's test for albumoses, 503 

for urobilin, 503 
Barfoed's reagent, 245 
Barfurth's reagent, 138 
Basic anilin dyes, 119 
double stain, 136 
phosphate of magnesium, 593 
Basophilic leucocytes in the blood, 80 
in the sputum, 348 
perinuclear granules, 78 
Baumann and v. Udranszky's method of 

isolating diamins, 583 
Beckmann's apparatus, 164 
Bence Jones' albumin, 487 



INDEX. 



681 



Bence Jones' albumin, tests for, 504 
Benzoic acid in the urine, 457 
Benzopurpurin test for hydrochloric acid, 

228 < 
Bile-pigment in the blood, 53 

in the feces, 333 

in the gastric contents, 261 

in the urine, 548 
tests for, 550 

Gmelin's, 550 
Huppert's, 550 
Kosenbach's, 550 
Smith's, 550 
Bilharzia haematobia, 195 

eggs in the urine, 629 
Bilharziasis, 195 
Biliary acids in the blood, 53 
in the feces, 283 
in the urine, 550 
tests for, 283 

concretions, 291 
analysis of, 292 
Bilirubin, 548, 599 
Biuret test, 423 
Blood, 17 

acetone in, 54 

albumins in, 27, 43 

albumoses in, 43 

alkalinity of, 20 

ammonia in, 47 

bacteriology of, 165 

biliary constituents in, 53 

carbohydrates in, 44 

cellulose in, 47 

chemical examination of, 27 

coagulation of, 28 

color of, 1 7 

color-index, 57 

crisis, 69 

drying and staining of, 115 

dust, 114 

examination, technique of, 114 

fat in, 50 

fatty acids in, 50 

fibrin in, 27, 43 

gases in, 31 

general characteristics of, 17 
chemistry of, 27 

glycogen in, 46 

hsemokonia of, 114 

in the feces, 274, 287, 296 

in the gastric contents, 261 

in the sputum, 340, 348 

in the urine, 413, 610 

kryoscopy of, 163 

lactic acid in, 52 

leucocytes of, 17, 71 

medico-legal test for, 39 

methods of staining, 123 

microscopical examination of, 54 

nucleated corpuscles in, 67 

odor of, 18 

parasites in, 165 



Blood, parasitology of, 165 
peptone in, 44 
pigments of, 32 
plaques, 17, 112 
proteids in, 43 
protozoa in, 182 
reaction of, 21 
red corpuscles, 17, 54 
solids of, 20 
specific gravity of, 18 
staining of, 119 
sugar in, 44 
tests for, 39, 507 

Donogany's, 262 
guaiacum, 507 
Heller's, 507 

Korczynski and Jaworski's, 288 
Muller and Weber's, 261 
urea in, 47 
uric acid in, 48 
xanthin-bases in, 49 
Blood-corpuscles, red, 54 

anaemic degeneration of, 62 
behavior toward anilin dyes, 62 
crenation of, 56 
granular degeneration of, 65 
enumeration of, 139 
money roll formation, 56 
nucleated, 67 
polychromatophilia of, 62 
variations in color, 56 
in form, 55 
in number, 58 
in size, 54 
white. See Leucocytes. 
Blood-crisis, 69 
Blood-iron, 158 
Blood-pigments, 32 
Blood-plasma, 17, 30 
Blood-plates, 112 
Blood-serum, 28 
Blood-shadows, 610 
Boas' bulbed stomach-tube, 213 

method for estimating lactic acid, 251 
test for hydrochloric acid, 227 
for lactic acid, 250 
Boas-Oppler bacillus, 264 
Bodo urinarius, 620 
Boiling test for albumin, 496 
Bothriocephalus latus, 311 
Boucher's crystals, 364, 661 
Bottger's test for sugar, 518 
Bremer's diabetic blood test, 63 

urine test, 530 
Brodie and Russell's method of enumerat- 
ing the plaques, 147 
Browning's spectroscope, 42 
Bubonic plague, bacillus of, 181 
Buccal secretion (see Saliva), 198 
Butyric acid fermentation, 254 
in the feces, 281 
in the gastric contents, 254 
test for" 254 



682 



INDEX. 



CABOT'S ring bodies, 67 
Cadaverin, 582 
Cahn-Mehring's method of estimating 

fatty acids, 255 
Calcium carbonate, crystals of, 601 
oxalate, crystals of, 292, 590 
phosphate, crystals of, 600 
sulphate, crystals of, 593 
Calomel stools, 271 
Carbohydrates, digestion of, 242 
in the blood, 44 
in the feces, 333 
in the urine, 503 
tests for, 243 
Carbol-fuchsin, 357 
Carbolic acid, estimation of, 567 

test for, 280, 567 
Carbolo-chloride of iron test for lactic 

acid, 248 
Carbon dioxide haemoglobin, 38 

monoxide haemoglobin, 37 
Caries of the teeth, 198 
Casein, digestion of, 242 
in the milk, 678 
test for, Leiner's, 295 
Casts, classification of, 612 
examination of, 613 
fatty, 616 
fibrinous, 342 
formation of, 618 
granular, 615 
hyaline, 614 
pus, 615 

significance of, 619 
staining of, 614 
urinary, 612 
waxy, 616 
Cause's method of estimating sugar, 524 
Cellulose in the blood, 47 
Cenomonadina, 301 
Cercomonas intestinalis, 301 
Cerebrospinal fluid, 649 
amount of, 650 
appearance of, 650 
chemical composition of, 652 
cytodiagnosis of, 653 
microscopical examination of, 

653 
reaction of, 652 
specific gravity of, 651 
toxicity of, 656 
Cestodes, 232 
Chalcosis, 366 

Charcot-Leyden crystals, in the feces, 275 
in the nasal discharge, 337 
in the sputum, 345, 364 
Chemical examination of blood, 27 
of cystic fluids, 657 
of exudates, 638 
of feces, 278, 333 
of gastric juice, 216 
of milk, 677 
of pus, 640 



Chemical examination of saliva, 198 
of semen, 661 
of sputum, 366 
of transudates, 633 
of urine, 385 
Chenzinsky-Plehn stain, 137 
Chlorides in the urine, 387 
estimation of, 390 

according to Neubauer and 

Salkowski, 395 
according to Salkowski and 

Volhard, 390 
direct method, 394 
test for, 390 
Chloroform-benzol mixture, 19 
Cholsemia, 53 
Cholera Asiatica, bacillus of, 323 

infantum, bacillus of, 331 
Cholesterin in the blood, 31 
in the feces, 282 
in the sputum, 365 
in the urine, 551 
isolation of, from the feces, 282 
test for, 283 
Choluria, 549 
Chorion villi, 670 
Chromogens in the urine, 537 
Chyluria, 579, 600 
Chymosin, 239 

estimation of, 241 
test for, 240 
Chymosinogen, 239 
estimation of, 241 
test for, 240 
Ciliated epithelium in cysts, 658 

in the sputum, 348 
Cladothrix, 362 
Coagulation of the blood, 28 
Coagulometer, Wright's, 29 
Coating of the tongue, 204 
Coffin-lid crystals, 591 
Colloid concretions in ovarian cysts, 658 
Color index of the blood, 57 
Colostrum, 672 
Comma bacillus, 323 
Concretions, biliary, 291 
fecal, 292 
intestinal, 291 
pulmonary, 346 
Congo-red test for free acids, 225 
Conjugate glucuronates, 533 
sulphates, 336, 555, 566 
Copper test for uric acid, 449 
Coproliths, 292 
Corpora amylacea, 663 
Crotonic acid, 576 

Crystals, ammonio-magnesium phos- 
phate, 591 
bilirubin, 599 
calcium carbonate, 601 
oxalate, 365 
phosphate, 592, 593 
sulphate, 593 



INDEX. 



<o8Z 



Crystals, Charcot-Leyden, 297 

cholesterin, 365 

cystin, 594 

fatty acids, 365 

hseniatoidin, 599 

hsemin, 39 

hippuric acid, 593 

indigo, 601 

in the feces, 274, 297 

leucin, 365 

leucocytic, 364 

magnesium phosphate, 593, 600 

monocalcium phosphate, 592 

neutral calcium phosphate, 593 

phenyl-glucosazon, 520 

phosphate of spermin, 661 

Teichmann, 39 

triple phosphate, 365 

tyrosin, 365 

urate of ammonium, 601 

uric acid, 588 

xanthin, 598 
Curschmann's spirals, 344 
Cylinders, mucous, in the feces, 290 
in the urine, 618 

urinarv, 612 
Cylindroids, 618 
Cylindruria, 612 
Cy stein, 411 

Cysticercus cellusosae, 308 
Cvstin, 412, 594 
Cystinuria, 412, 582, 594 
Cysts, colloid, 653 

contents of, 657 

dermoid, 658 

fibrocystic, 658 

hydatid, 659 

ovarian, 659 

pancreatic, 660 

parovarian, 658 
Cytodiagnosis in cerebrospinal fluid, 
653 

in pleural effusions, 635 



D ALAND'S hamiatokrit, 147 
Dare's hremoalkalimeter, 25 
haernoglobinorneter, 151 
method of estimating the alka- 
linity of the blood, 25 
Decidual cells, 671 
Deetjen's agar, 112 
cf-granulation of Ehrlich, 80 
Dennige's test for acetone, 54, 570 
Dermoid cysts, 658 
Dextrin in the urine, 532 
Dextrose in the urine. See Glucose. 
Diabetes, 515 

alternans, 447 

Bremer's blood test in, 63 

urine test in, 530 
hepatogenic, 516 
Hirschfeld's form of, 517 



Diabetes, insipidus, 374, 534 
myogenic, 516 
phosphatic, 398 
Williamson's blood test in, 46 
Diabetic chromatophilia, 63 
Diacetic acid in the urine, 573 

tests for, 573 
Diaceturia, 573 
Diamins in the feces, 334 
in the urine, 582 

isolation of, 583 
Diarrhoea, 285 _ 
Diathesis, oxalic acid, 466 

uric acid, 446 
Diazo-reaction (see Ehrlich's reaction), 

559 
Differential density method of estimating 

sugar, 526 
Digestion, gastric, 241 

of albumins, 241 
of albuminoids, 242 
of carbohydrates, 242 
of milk, 242 
of proteids, 242 
products of, 241 
Dimethylamidoazobenzol test, 226 
Diphtheria, 206 

Diplococcus meningitidis intracellularis, 
654 
pneumoniae, 360 

in the blood, 171 
Dipylidium caninum, 309 
Distoma Buskii, 314 
capense, 195 
conjunctum, 315 
haematobium, 195, 315, 354 
hepaticum, 313 
heterophyes, 315 
lanceolatum, 314 
pulmonale, 315, 353 
rhatonisi, 314 
sibiricum, 314 
spatulatum, 314 
Distomiasis, 195 
Donne's pus test, 607 
Donogany's blood test, 262, 507 
Doremus' ureometer, 428 
Drugs, effect of, on the color of the stools, 

271 
Drysdale's corpuscles, 659 
Dunlop's method of estimating oxalic 

acid, 471 
Dust-particles of Miiller, 114 
Dyes, 119 



EARTHY phosphates, 395 
Eberth's bacillus, 325 
Echinococcus, 345, 352 

membranes in the sputum, 345 
polymorph us, 352 
e-granulation of Ehrlich, 77 
Ehrlich's dahlia, 136 



684 



INDEX. 



Elirlich's diazo-reaetion, 559 

dimethylamidobenzaldehyde reac- 
tion, 564 
eosin-methylene-blue methylal, 133 
haematoxylin-eosin, 138 
hsemoglobinaemic Innenkorper, 67 
methyl-green-fuchsin stain, 136 
neutral red, 645 
tri-acid stain, 124 
tri-glycerin mixture, 135 
Einhorn's bucket, 215 

saccharimeter, 519, 526 
Elastic tissue in the sputum, 342, 350 

stain for, 351 
Eisner's method, 327 
Engel's alkalimeter, 25 

method of estimating the alkalinity 
of the blood, 24 
Enterogenic albumosuria, 486 
Enteroliths, 292 
Eosin, staining with, 135 
Eosin-methylal and methylene-blue, 133 
Eosinate of methvlene-blue, staining with, 

123 
Eosinophilia, 100 

Eosinophilic leucocytes in the blood, 
79 
in the sputum, 347 
Epithelial cells, alveolar, 349 
ciliated, 349 

in the buccal secretion, 200 
in the feces, 274, 295 
in the gastric contents, 201 
in the" sputum, 348 
in the urine, 602 
in the vaginal secretions, 666 
Eructatio nervosa, 258 
Erythrodextrin, 242 

test for, 242 
Erythrosin, acid, staining with, 139 
Esbach's albuminimeter, 500 

method of estimating albumin, 500 
reagent, 500 
Escherich's stain, 329 
Ethyl sulphide, 411 
Euchlorhydria, 224 
Eustrongylus gigas, 629 
Ewald's modification of Mohr's test for 

hydrochloric acid, 228 
Ewing's stain, 132 
Extractives in the blood, 31 
Exudates, 634 

bacteriological examination of, 639 

chemistry of, 638 

chyloid, 647 

chylous, 647 

hemorrhagic, 634 

in cancer, 636 

in tuberculosis, 635 

purulent (see Pus), 640 

putrid, 649 

serous, 634 



FAKRANT'S solution, 614 
Fat in the blood, 50 

in the milk, estimation, 677 

in the urine, 578, 599 
Fatty acids, clinical significance of, 253 

estimation of, 255 

formation of, 253 

in pus, 641 

in the blood, 50 

in the feces, 280 

in the gastric contents, 253 

in the sputum, 363 

in the urine, 577 

tests for, 253 
casts, 616 
Febrile acetonuria, 468 
albuminuria, 477 
urobilin, 552 
Fecal matter in the urine, 630 

vomiting, 262 
Feces, 270 

alimentary detritus in, 272, 289 
amount of, 270, 286 
annelides in, 315 
biliary acids in, 282 

concretions in, 227 
blood in, 287, 296 
chemistry of, 278, 333 
cholesterin in, 282 
color of, 271, 287 
composition of, 210 
concretions in, 291 
consistence of, 271, 285 
crystals in, 274, 297 
epithelial cells in, 274, 295 
examination of normal, 207 
fatty acids in, 280 
flagellata in, 300 
foreign bodies in, 272 
form of, 271, 285 
gases in, 279 

general characteristics of, 270, 285 
indol in, 279 
leucocytes in, 274, 296 
macroscopical constituents of. 272, 

289 
microscopical constituents of, 272, 

293 
mucus in, 290, 296 
number of stools, 270, 285 
odor of, 271, 286 
parasites in, 275, 297, 323 

animal, 297 

vegetable, 275, 323 
pathology of, 285 
phenol in, 279 
pigments in, 283 
protozoa in, 298 
ptomai'ns in, 334 
reaction of, 278, 286 
skatol in, 279 

technique in examination of, 293 
trematodes in, 313 



INDEX. 



685 



Feces, vermes in, 305 

Fehling's method of estimating sugar, 523 

solution, 518 

test for sugar, 517 
Ferment, milk-curdling, 239 

of saliva, 199 
Fermentation test for sugar, 519 

Schmidt's fecal, 293 
Ferments in the gastric juice, 236 

in the urine, 579 
Ferrocyanide test for albumin, 497 
Ferrometer, Jolles', 158 
Feser's lactoscope, 677 
Fibrin, 28, 43 

estimation of, 43 

ferment, 28 

in the blood, 28 

in the urine, 490 

test for, 508 
Fibrinogen, 28 
Fibrinoglobulin, 28 
Fibrinous casts, 342 

coagula in the sputum, 342 

in the urine. See Chyluria. 
Filaria Bancrofti, 192 

demarquaii, 192 

diurna, 192 

Mansoni, 192 

nocturna, 192 

ozzardi, 192 

perstans, 195 

sanguinis hominis, 192 

Wuchereri, 192 
Filariasis, 192 
Finkler-Prior bacillus, 325 
Flagellata, 300 
Fleischl's hsemometer, 153 
Florence's test for semen, 664 
Folin's method of estimating ammonia, 
441 
kreatinin, 464 
urea, 434 
uric acid, 450 
Foreign bodies in the feces, 272 
in the sputum, 346 
in the urine, 630 
Formic acid, detection of, 281 
Freund's method of determining acidity 

of urine, 384 
Furfurol test for bile acids, 283 
Futcher's stain, 137 

GABETT'S staining method, 357 
Galacturia, 599 
Gall-stones, analysis of, 292 

in the feces, 291 
Garrod's test for hsematoporphyrin in the 
urine, 547 
for homogentisinic acid, 558 
for uric acid in the blood, 49 
Gases in the blood, 31 
in the feces, 279 
in the gastric contents, 256 



Gases in the urine, 580 
Gastric contents, examination of (see 
Gastric juice), 210 
digestion of albuminoids, 242 
of carbohydrates, 242 
of native albumins, 241 
of proteids, 242 
products of, 241 
analysis of, 244 
juice, 210 

acetic acid in, 253 
acetone in, 258 
acidity of, 217, 219 
amount of, 215 
antiseptic properties of, 222 
blood in, 261 
butyric acid in, 253 
cause of acidity of, 217 
chemical composition of, 216 

examination of, 216 
chymosin in, 239 
chymosinogen in, 239 
fatty acids in, 253 
ferments in, 236 
free acid in, 225 
gases in, 256 

general characteristics of, 215 
hydrochloric acid in, 217, 224 
hyperacidity of, 221 
hypersecretion of, 221 
lactic acid in, 245 
methods of obtaining, 213 
microscopical examination of, 

264 
milk-curdling ferment of, 239 
organic acids in, 253 
pepsin in, 236 
pepsinogen in, 236 
ptomains and toxalbumins in, 
258 
• secretion of, 210 
zymogens in, 236 
Gastrosuccorrhoea mucosa, 260 
Gerhardt's test for diacetic acid, 573 

for urobilin, 553 
Giemsa's stain, 129 
Gigantoblasts (see Megaloblasts), 69 
Glanders, bacillus of, 178 
Glandular fever, 206 
Glaser's method of estimating neutral 

sulphur, 413 
Globin, 32 
Glucose, 508 

in the blood, 44 

estimation of, 45 
in the urine, 508 
quantitative estimation of, 522 
tests for, 517 
Glucosuria, 508 
digestive, 508 
e saccharo, 512 
ex amylo, 512 
persistent, 514 



686 



INDEX. 



Glucosuria, transitory, 513 
Glucosuric acid, 555 
Glucuronic acid in the blood, 44 

in the urine, 533 

Glycogen in the blood, 46 

test for, 138 

in the sputum, 366 
Gmelin's reaction, 550 
Gonococcus in the blood, 176 

in. the mouth, 203 

in the urine, 627 
staining of, 645 

in urethral discharge, 644 

Neisser's, 644 
Gonorrheal pus, 644 

stomatitis, 203 

threads in the urine, 608, 646 
Gowers' haemoglobinometer, 157 
Gram's method of staining, 207 
Granular degeneration, 65 
^-granulation of Ehrlich, 80 
Grape-sugar. See Glucose. 
Green's ureometer, 432 
Gregarina, 305 

Grethe's method of staining tubercle ba- 
cilli in the urine, 626 
Guaiacum test for blood, 507 
Guanin in the urine, 507 
Gum, animal, 533 
Gunning's mixture, 435 
Giinzburg's packages, 268 

reagent, 226 
Gynaecophorus, 195 

HJEMATEMESIS, 262 
Haematin, 38 
Haematin uria, 489, 545 
Haematoblasts, 112 
Haematoidin in the blood, 40 
in the sputum, 364 
in the urine, 599 
Haematokrit, 147 

Haematoporphyrin in the blood, 41 
in the feces, 284 
in the urine, 546 
tests for, 547 
Haematoporphyrinuria, 546 
Hematuria, 490, 610 
Haemin (see Teichmann's crystals), 39 
Haemo-alkalimeter, Dare's, 26 
Haemochromogen, 32 
Haemocytometer of Thoma-Zeiss, 139 
Haemoglobin, 17, 32 
carbon dioxide, 38 
monoxide, 37 
estimation of, with Dare's instru- 
ment, 151 
with Fleischl's haemometer, 153 
with Gowers' haemoglobinome- 
ter, 157 
with Oliver's instrument, 155 
with Talquist's method, 158 
hydrogen sulphide, 38 



Haemoglobin, nitric oxide, 38 

tests for, 39, 506 
Haemoglobinaemia, 36 
Haemoglobinometer, 150 
Haemoglobinuria, 36, 489 
Haemokonia, 114 
Haemometers, 150 
Haemospermia, 663 
Halitus sanguinis, 18 
Hammerschlag's method of determining 
the specific gravity of blood, 
19 
of estimating pepsin, 238 
Haycraft's method of estimating uric acid, 

452 
Hayem's fluid, 141 
Heart-disease cells, 350 
Hehner-Seemann's method of estimating 

organic acids, 255 
Heller's test for albumin, 493 

for blood, 507 
Hepatogenic icterus, 549 
Heteroxanthin in the urine, 455 
Hippuric acid in the urine, 457, 593 
estimation of, 459 
properties of, 458 
test for, 458 
Histon in the urine, 492 

test for, 508 
Hoffmann's test for tyrosin, 597 
Hofmeister's method of estimating hip- 
puric acid, 460 
test for leucin, 598 
Homogentisinic acid in the urine, 555 
estimation of, 558 
isolation of, 557 
Hopkins' method of estimating uric acid, 

449 
Hufner's ureometer, 432 
Huppert's test for bile-pigment, 550 
Hydatid cysts, 659 

echinococcus membranes and 

hooklets in, 659 
sodium chloride in, 659 
succinic acid in, 659 
disease, 352 
Hydrobilirubin, 284 
Hydrocele agar, 646 
fluid, 633 

cholesterin in, 633 
Hydrochinon in the urine, 567 
Hydrochloric acid in the gastric juice, 217 
amount of, 224 
combined, 229 

estimation of, according to Leo, 
235 ^ 
according to Martius and 

Liittke, 232 
according to Sahli, 232 
according to Topfer, 230 
free, 224 

significance of, 222 
source of, 221 






INDEX. 



687 



Hydrochloric acid, tests for, 225 
Hydrogen sulphide, in the gastric con- 
tents, 257 
tests for, 257 
in the urine, 580 
Hydronephrosis, 659 
Hydrothionuria, 342, 507, 580, 627 
Hymenolepis, 308 
.Hypalbuminosis, 43 
Hypeosinophilia, 104 
Hyperalbuminosis, 43 
Hyperchlorhydria, 225 
Hyperinosis, 43 
Hyperisotonia, 31 
Hyperleucocytosis, 85 

polynuclear eosinophilic, 100 
neutrophilic, 86 
Hvpersecretio acida et continua, 221, 225 
Hypersecretion, 216, 221 
Hypinosis, 43 

Hypobromite method of estimating urea, 
426 
solution, 426 
Hypochlorhydria, 224 
Hypoleucocytosis, 85 

polynuclear eosinophilic, 104 
neutrophilic, 96 
Hypoxanthin in the urine, 455 

TCTERUS, 549 

_L hematogenic, 549 

hepatogenic, 549 

neonatorum, 549 

urobilin, 552 
Idiopathic bacteriuria, 627 

oxaluria, 468 
Ilasvay's reagent, 200 
Indican in the urine, 537 
estimation of, 541 
tests for, 540 
Indicanuria, 537 

Indigo-blue in the urine, 559, 581, 601 
Indigo-red in the urine, 543 
Indigosuria, 559, 581, 601 
Indol in the feces, 279 
tests for, 280 
Indoxyl, 537 

sulphate (see Indican), 537 
Influenza, bacillus of, 178 
Infusoria in pus, 643 

in the feces, 297 

in the urine, 629 

in vaginal discharges, 667 
Inosit in the urine, 534 
Insects in the feces, 323 
Intermittent albuminuria, 474 
Intestinal concretions, 292 

sand, 292 
Iodoform-test for lactic acid, 250 
Iodophilia, 83 

demonstration of, 138 
Iodospermin, 664 
Iron in blood, 36 



Isomastigoda, 300 
Isotonia, 30 

JAFFE'S test for indican, 541 
Jaundice (see Icterus), 549 
Jenner's stain, 123 
Jolles' ferrometer, 158 
Justus' syphilitic blood test, 35 

KELLING'S test for lactic acid, 249 
Kjeldahl's method, 435 
Knapp's method of estimating sugar, 525 
Koch's stain, 132 
Koplik's bacillus, 361 
Korczynski and Jaworski's test, 288 
Krabbea grandis, 313 
Kreatin, 460 

properties of, 461 
Kreatinin, 460 

estimation of, 462 

properties of, 461 

test for, 462 
Kreatinin-zinc chloride, 462 
Kryoscopy of the blood, 163 

of the urine, 585 

LACMOID paper, preparation of, 24 
Lactic acid, 245 

clinical significance of, 245 
estimation of, 251 
fermentation, 248 
in the blood, 52 
in the gastric contents, 245 
in the urine, 576 
mode of formation, 245 
tests for, 248 
Boas', 250 
Killing's, 249 
Strauss', 249 
Uffelmann's, 248 
Lactodensimeter of Quevenne, 675 
Lactoscope of Feser, 677 
Lactose in the urine, 531 
Lsevulose in the urine, 531 
Laiose in the urine, 532 
Landois' estimation of the alkalinity of 

the blood, 21 
Large mononuclear leucocytes, 75 

clinical variations of, 107 
Laveran's organism, 182 
Laverania malariae, 187 
Lecithin in the blood, 31 
Legal's test for acetone, 569 
Leiner's test for casein, 295 
Leishman's stain, 130 
Leo's method of estimating hydrochloric 

acid, 235 
Leprosy, bacillus of, 356 
Leptothrix buccalis, 200 
Leube's test of motor power of stomach, 

267 
Leucin, 595 
Leucocytes, 17, 71 



688 



INDEX. 



Leucocytes, basophilic, 80 

degenerative changes, 77, 641 

differential enumeration, 146 

differentiation according to their be- 
havior toward anilin dyes, 73 

Ehrlich's granulations in, 77, 79, 80 

enumeration of, 143 

eosinophilic, 79 

estimation of the number of, 143 

general differentiation of the various 
forms, 71 

indirect enumeration of, 145 

in the blood, 17, 71 

in the exudates, 635 

in the feces, 274, 296 

in the sputum, 346 

in the urine, 605 

irritation forms, 83 

large mononuclear, 75 

lymphogenic, 73 

myelocytes, 81 

neutrophilic, 77 

oxyphilic, 79 

polymorphonuclear, 72, 77 

polynuclear, 72, 77 

pseudolymphocytes, 83 

small mononuclear, 73 

transition forms, 75 

variations in number of, 85 
Leucocytic crystals, 364 
Leucocytosis (see Hyperleucocytosis), 85 
Leucopenia, 85, 96 
Leukaemia, lymphatic, 107 

myelogenous, 109 
Lieben's test for acetone, 570 
Lientery, 289 
Lipacichemia, 50 
Lipaciduria, 577 
Lipa?mia, 50 
Lipuria, 578, 599 
Lochia, 668 

alba, 668 

rubra, 668 
Loftier' s bacillus, 206 

methylene-blue solution, 206 
Lohnstein's saccharimeter, 527 
Lowy's method of estimating the alka- 
linity of the blood, 24 
Ludwig-Salkowski's method of estimating 

uric acid, 453 
Lymphocytes, 73 
Lymphocytosis, 75, 105 
Lymphopenia, 75, 107 

MACEOCYTES, 54 
Macrocythsemia, 55 
Magnesia mixture, 401 

soaps of, in the urine, 598 
Magnesium phosphate, 593, 600 
Malaria, plasmodium of, 182 
Malta fever, bacillus of, 180 
Maltose in the urine, 531 
Mammary secretion, 672 



Marcano's fluid, 142 
Marrow cells, 82 
Marshall's ureometer, 432 
Marsh gas in the gastric contents, 256 
Martius and Liittke's method of esti- 
mating hydrochloric acid, 232 
Masons' lung (see Siderosis), 365 
Mast cells, 80 

clinical variations of, 108 

stains for, 136 
Meconium, 334 
Medico-legal test for blood, 39 
Megaloblasts, 69 
Megalocytes, 54 
Megastoma entericum, in the feces, 303 

in the gastric contents, 263 
Melamernia, 554 
Melanin in the urine, 554 

tests for, 554 
Melanogen, 554 

Membranous dysmenorrhea, vaginal dis- 
charge in, 669 
Meningeal fluid, examination of, 649 
Menstruation, vaginal discharge in, 668 
Metalbumin in ovarian cysts, 657 
Methsemoglobin, 40 

sulphide, 38 
Methemoglobinemia, 37, 40 
Methpemoglobinuria, 489 
Methane (see Marsh gas), 256 
Methylene azure, 137 
Mett's method of estimating pepsin, 238 
Michaelis' stain, 134 
Michaelis and Wolff's stain, 129 
Microblasts, 70 
Micrococci in pus, 642 
Micrococcus urea?, 624 
Microcytes, 54 
Microcythsemia, 55 
Micro-organisms, in pus, 642 
in the feces, 276, 323 
in the milk, 675 
in the mouth, 200 
in the nasal secretion, 336 
in the urine, 626 
in vaginal discharges, 667 
Microscopical examination of cystic fluids, 
658 

of exudates, 635 

of the blood, 54 

of the buccal secretion, 200 

of the feces, 272, 293 

of the gastric contents, 264 

of the nasal secretion, 336 

of the sputum, 346 

of the urine, 585 

of the vaginal secretion, 666 

of the vomitus, 262 

of transudates, 634 
Miescher's hsemometer, 155 
Milk, 672 

^hemical composition of, 673 
cows', 673 



INDEX. 



689 



Milk, examination of, 677 

fat in, estimation of, 677 

human, 673 

in disease, 674 

proteids of, 678 

secretion of, in the adult female, 672 
in the newly born, 672 

specific gravity of, 673 

witches', 672 
Milk-curdling ferment in the gastric 

juice, 239 
Millon's reagent, 502 
Mohr's test for hydrochloric acid, 228 
Monadina in the feces, 300 
Monera in the feces, 298 
Monocalcium phosphate, 592 
Moro's bacillus, 329 

Motor power of stomach, examination of, 
266 
Leube's method, 267 
salol test of Ewald and Sievers, 
267 
Moulds in the urine, 628 
Mouth, actinomycosis of, 203 

secretions of, 198 

tuberculosis of, 203 
Mucin in the feces, 333 

in the urine, 491 
test for, 505 
Mucous corpuscles in the urine, 369 

cylinders in the feces, 290 
in the urine, 618 
Mucus in the feces, 290, 296 

in the gastric contents, 260 
Muller-Weber test for blood, 261 
Murexid test, 449 
Myelsemia, 86, 109 
Myelin granules in the sputum, 349 
Myelocytes, basophilic, 83 

eosinophilic, 82 

neutrophilic, 82 
Myelocytosis, 86, 109 

NASAL catarrh, 336 
secretion, 336 

cerebrospinal fluid in, 336 

characteristics of, 336 

Charcot - Leyden crystals in, 

337 
in disease, 336 
Neisser, gonococcus of, 644 
Nematodes, 315 
Nessler's reagent, 250 
Neubauer's method of estimating oxalic 

acid, 470 
Neusser's granules, 78 
Neutral dyes, 121 

phosphate of calcium in the urine, 

593 # 
red stain for gonococci, 645 
sulphur in urine, 411 
Neutrophilic leucocytes, 77 
Nitric acid test for albumin, 493 
44 



Nitric oxide haemoglobin, 38 
Nitrites in the saliva, 199 
Nitrogen in the urine, 418 
estimation of, 435 

according to Kjeldahl, 435 
according to Will-Varren- 
trapp, 437 
Nitrogenous equilibrium, 418 
Nitro-prusside of sodium as a test for 

acetone {see Legal's test), 569 
Nocht-Romanowsky stain, 127 
Normal urobilin, 535 
Normoblasts, 68 
Nose, secretion from, 336 
Nubecula in the urine, 369 
Nucleated red corpuscles, 67 
Nucleo-albumin in the blood, 43 
in the urine, 491 
test for, 505 
Nucleohiston in the urine, 492 
Nummular sputum, 341 
Nylander's test for sugar, 518 

OBERMAYER'S reagent, 540 
Obermeier, spirochseta of, 179 
Oi'dium albicans, 204, 363 
Olefiant gas, 257 
Oligochromsemia, 33 
Oligocythemia, 34, 59 
Oliguria, 375 

Oliver's haemoglobin ometer, 155 
Orcin test for pentoses, 532 
Organic acids in the blood, 50 

in the gastric juice, 253 

quantitative estimation 
of, 255 

in the sputum, 363 
Organized sediments of the urine, 602 
Ott's test, 506 
Ovarian cysts, 654 

Oxalate of calcium crystals in the sputum, 
363 
in the urine, 590 
Oxalic acid, diathesis, 466 

in the urine, 466 

properties of, 469 

quantitative estimation of, 470 

tests for, 469 
Oxaluria idiopathica, 468 
Oxaluric acid, 466 
Oxyamygdalic acid, 577 
Oxybutyric acid, /3, in the urine, 574 

estimation of, 575 
Oxyhemoglobin, 18, 32 
Oxyphilic leucocytes, 79 
Oxyuris vermicularis, 316 
Ozsena, 337 

PACINI'S fluid, 142 
Pancreatic cysts, 660 

trypsin in, 660 
juice in the gastric contents, 261 
Pappenheim's methyl-green-pyronin, 136 



690 



INDEX. 



Pappenheim's stain for tubercle bacilli, 
357 
tri-acid stains, 126 
imoecium c 
Paramucin, 65' 
Parasites in the blood, 182 
in the feces, 275, 297, 323 
in the gastric contents, 263 
in the sputum, 352 
in the urine, 623 
malarial, 182 
Paratyphoid fever, bacilli of, 170, 328 
Paraxanthin in the urine, 442, 455 
Patein's albumin, 485 
test for, 499 
Pathological acetonuria, 568 
albuminuria, 472 
glucosuria, 508 
urobilin, 535, 551 
Pentoses in the urine, 532 

_ tests for, 532 
Pepsin in the gastric ."juice, 236 
estimation of, 238 
\ tests for, 238 
Pepsinogen in the gastric juice, 236 
estimation of, 239 
tests for, 238 
Peptones in the blood, 44 
in the feces, 333 
in the urine, 489 
test for, 504 
Peptonuria, 489 
Persistent glucosuria, 514 
Petrone's fluid, 142 
Pettenkofer's test, 283 
Phagocytes, 72 
Phagocytosis, 72, 190 
Pharyngomycosis leptothrica, 205 
Phenol," 21 6, 555, 566 
estimation of, 567 
in the feces, 279 
in the urine, 555, 566 
tests for, 216, 566 
Phenylglucosazon, 520 
Phenylhydrazin test for sugar, 520 
Phloroglucin test for pentoses, 533 

vanillin test for hydrochloric acid, 
226 
Phosphates in the urine, 395 
estimation of, 401 
removal of, from urine, 404 
separate estimation of alkaline 

and earthy, 404 
tests for, 400 
Phosphatic diabetes, 398 

sediments in the urine, 591 
Picric acid test for albumin, 493 
Pigments in the feces, 283 

in the urine, 534 
Piorkowski's method, 326 
Piria's test for tyrosin, 597 
Placenta sanguinis, 28 
Plague bacillus, 362 



Plaques, 112 

enumeration of, 147 
Plasma of the blood, 17, 24 
Plasmodium malaria?, 182 

crescentic bodies, 187 
flagellate bodies, 188 
hyaline bodies, 183 
ovoid bodies, 187 
pigmented extracellular bodies, 
188 
intracellular bodies, 184 
segmenting bodies, 186 
spherical bodies, 187 
staining of, 183 
Platodes, 297 
Plehn's stain, 137 
Pneumoconioses, 365 
Pneumonia, diplococcus of, 360 
in the blood, 171 
sputum in, 360 
Poikilocytes, 55 
Poikilocytosis, 55 
Polarimeter, 528 
Pole bacillus, 361 
Polychromasia, 62 
Polychromatophilic degeneration, 62 
Polycythemia, 58 
Polymastigina, 301 
Polyuria, 372 
Propepsin, 236 
Prostatic fluid, 661 
Proteids, formed in the stomach, 242 
of the blood, 43 
reaction of, 244 
Proteus vulgaris, 330 
Protozoa, 298 
in pus, 643 
in the blood, 182 
in the feces, 298 
in the sputum, 352 
in the urine, 623 
Pseudocasts, 618 
Pseudogonococci, 646 
Pseudolymphocytes, 83 
Pseudomucin, 657 
Psorospermiasis, 305 
Ptomains in the feces, 334 

in the gastric contents 258 
in the urine, 581 
isolation of, 583 
Ptyalin, 199 

test for, 199 
Purin, 442 

bases, 442, 455 
Purulent exudates, 640 
Pus, 640 

chemistry of, 640 
corpuscles, in the urine, 605 
enumeration of, 609 
crystals in, 643 
detritus in, 642 
general characteristics of, 640 
giant corpuscles in, 642 



INDEX. 



691 



Pus, gonorrhoeal, 644 

in the feces, 296 

in the gastric contents, 262 

in the urine, 605 

leucocytes in, 641 

microscopical examination of, 641 

organisms in the urine, 625 

parasites in, 642 

protozoa in, 643 

red corpuscles in, 642 

tests for, 606 
Putrescin, 582 
Pyogenic albumosuria, 486 
Pyrocatechin in the urine, 567 
Pyrocatechuic acid, 555 
Pyroplasma hominis, 192 
Pyuria, 605 



Q' 



RAY fungus, 362 
Beach's test, 269 
Ked blood-corpuscles, 17, 54 

anaemic degeneration of, 62 
behavior toward anilin dyes, 62 
enumeration of, 139 
granular degeneration of, 65 
nucleated forms, 67 
variations in color, 56 
in form, 55 
in number, 58 
in size, 54 
Eelapsing fever, spirillum of, 179 
Renal albuminuria, 477, 484 
Resorcin test, 227 

Resorptive power of the stomach, ex- 
amination of, 268 
Reuters stain, 129 
Reynolds' test for acetone, 570 
Rhamnose in urine, 532 
Rhizopoda, 298 

Romanowsky method, of staining, 127 
Rosenbach's reaction, 544 

test for bile-pigments, 550 
Round worms, 315 
Roy's method of determining the specific 

gravity of the blood, 18 
Rust-colored expectoration, 340 

SACCHARIMETERof Einhorn, 519 
of Lohnstein, 527 
of Soleil-Ventzke, 528 
Saccharomyces cerevisise. See Yeast. 
Saliva, 198 

chemistry of, 198 
f general characteristics of, 198 
in special diseases of the mouth, 203 
in the gastric contents, 261 
microscopical examination of, 200 
nitrites in, 199 
pathological alterations of, 202 



Saliva, ptyalin in, 199 

salivary corpuscles in, 200 
test for nitrites, 199 
for ptyalin, 199 
for sulphocyanides, 199 
Salivation, 202 

Salkowski's method of estimating oxalic 
acid in urine, 471 
xanthin-bases in urine, 456 
test for albumoses, 502 
for phenol, 566 
Salkowski-Neubauer method of estimat- 
ing the chlorides in urine, 395 
Salkowski-Volhard method of estimating 

the chlorides in urine, 390 
Salol test of Ewald and Sievers, 267 
Sanarelli's Bacillus icteroides, 180 
Sand, intestinal, 292 
Sarcina pulmonalis, 363 
urinae, 628 
ventriculi, 265 
Scarlatina, pharyngeal secretion in, 209 
Schauffler's methylene-blue-pyronin, 207 
Scherer's test for leucin, 597 
Schistosoma, 195 
Schizomycetes in the feces, 276 
Schlosing's method of estimating ammo- 
nia, 441 
Schmalz and Peiper's method of deter- 
mining the specific gravity of the blood, 
19 
Schmidt's fecal fermentation test, 293 
Secretions of the mouth, 198 
Sediments in acid urines, 588 
in alkaline urines, 600 
urinary, 369, 585 

ammonio-magnesium phosphate 

in, 591 
ammonium urate in, 601 
amorphous urates in, 589 
basic magnesium phosphate in, 

593 
bilirubin in, 599 
brick-dust, 588 
calcium carbonate in, 601 
oxalate in, 590 
sulphate in, 593 
cystin in, 594 
epithelial cells in, 602 
fat in, 599 

foreign bodies in, 630 
ha?matoidin in, 599 
hippuric acid in, 593 
indigo in, 601 
leucin in, 595 
leucocytes in, 605 
mode of examination of, 613 
monocalcium phosphate in, 592 
neutral calcium phosphate in, 

593 
non-organized, 588 
organized, 602 
red corpuscles in, 610 



692 



INDEX. 



Sediments, urinary, soaps of lime and 
magnesium in, 598 
spermatozoa in, 622 
tube-casts in, 612 
tumor particles in, 630 
tyrosin in, 595 
urates in, 589, 601 
uric acid in, 588 
parasites in, animal, 628 

vegetable, 623 
xanthin in, 598 
Seegen-Schneider method of estimating 

nitrogen, 437 
Semen, 661 

chemistry of, 661 
general characteristics of, 661 
microscopical examination of, 662 
pathology of, 663 
recognition of, in stains, 663 
spermatic crystals in, 661 
spermatozoa in, 662 
Sepsis, organisms in the blood in, 173 
Serosamucin, 638 
Serous exudates, 634 
Serum-albumin in the blood, 28 
in the urine, 472 

estimation of, 499 
tests for, 492 
Serum-globulin in the blood, 28 
in the urine, 485 

estimation of, 502 
test for, 502 
Shiga's bacillus, 331 
Siderosis, 365 
Skatol in the feces, 279 

tests for, 280 
Skatoxyl, 566 

sulphate, 566 
Sleeping sickness, organism of, 191 
Smegma bacillus, 356, 361, 626 
Soaps of lime and magnesium in the 

urine, 598 
Sodium chloride in hydatid fluid, 659 
Spectroscope, 41 
Spermatic crystals, 364 
Spermatocystitis, 623 
Spermatorrhoea, 623 
Spermatozoa in the semen, 662 

in the urine, 622 
Spermin, 661 
Spiegler's reagent, 498 
Spirals of Curschmann, 344 
Spirillum of relapsing fever, 179 
Spirochfeta Obermeieri, 179 
Sporozoa, 305 

Spotted fever, organism of, 192 
Sputum, 338 

Amoeba coli in, 353 
amount of, 339 
bacteria in, 354 
blood in, 348 
cheesy particles in, 342 
chemistry of, 366 



Sputum, color of, 340 

concretions in, 346 

configuration of, 341 

consistence of, 339 

crudum, 341 

crystals in, 363 

Curschmann's spirals in, 344 

Distoma pulmonale in, 283 

echinococcus in, 345 

elastic tissue in, 342, 350 

epithelial cells in, 348 

fibrinous casts in, 342 

foreign bodies in, 346 

general characteristics of, 339 

globosum, 341 

heterogeneous, 341 

homogeneous, 341 

leucocytes in, 346 

macroscopical constituents of, 342 

microscopical examination of, 346 

nummular, 341 

odor of, 340 

parasites, animal, in, 352 
vegetable, in, 354 

specific gravity of, 341 

technique in the examination of, 338 
Squibb's ureometer, 433 
Staining, methods of, 123 

principles of, 119 
Staphylococcus pyogenes albus, 176 
aureus, 175 
citreus, 176 
Starch, digestion of, 199 

solution, 252 
Steatorrhea, 289 
Stercobilin, 284 

Stercoraceous material in the vomit, 262 
Stock's test for acetone, 570 
Stokes' fluid, 33 
Stomach, motor power of, 266 

rate of absorption in, 268 

washing, 215 
Stomach-tube, 212 

contraindications to its use, 213 

its introduction, 213 
Stomatitis, catarrhal, 203 

gonorrhceal, 203 

ulcerative, 203 
Stools. See Feces. 
Strauss and Kohnstein's stain, 134 
Strauss' test for lactic acid, 249 
Streptococcus pyogenes, 176 
brevis, 176 
conglomeratus, 176 
longus, 176 
Streptothrix actinomycotica, 362 
Strongyloides, 320 
Strongylus duodenalis, 317 
Stycosis, 366 

Succinic acid in hydatid fluid, 659 
Sudan stain for fat, 600 
Sugar in the blood, 44 



INDEX. 



693 



Sugar in the urine, estimation of, 522 

tests for, 517 
Sulphanilic acid test. See Ehrlich's n 

tion. 
Sulphates in the urine, 404 
conjugate, 405 
estimation of, 409 
mineral, 405 
tests for, 408 
Sulphocyanides in the saliva, 199 

in the urine, 411 
Sulphur, neutral, in urine, 411 
estimation of, 413 
Syphilitic blood test of Justus, 35 



TAENIA africana, 310 
canina, 309 

cucumerina, 309 

diminuta, 309 

echinococcus, 352 

flavopunctata, 309 

madagascariensis, 311 

mediocanellata, 306 

nana, 308 

saginata, 306 

solium, 307 
Talquist's hsemoglobinometer, 158 
Tartar, 204 

Tauro-carbaminic acid in urine, 411 
Teichmann's crystals, 39 
Test-breakfast of Boas, 211 

of Ewald and Boas, 211 
Test-dinner of Biegel, 211 
Test-meal of Salzer, 211 
Test-meals, 211 
Thecosoma, 195 
Thiosulphates in urine, 44 
Thoma-Zeiss' haemocvtometer, 139 
Thrush, 204 
Toison's fluid, 141 
Tollen's orcin test, 532 

phloroglucin test, 533 
Tongue, coating of, 204 
Tonsillitis, 205 
Tonsils, coating of, 205 
Topfer's method of estimating hydro- 
chloric acid, 230 

test for hydrochloric acid, 226, 230 
Toxalbumins in the gastric contents, 

258 
Transitory glucosuria, 513 
Transudates, 631 

albumin in, 632 

chemistry of, 633 

coagulation of, 633 

general characteristics of, 631 

microscopical examination of, 634 

specific gravity of, 631 
Trematodes, "297 
Triacid stain, Ehrlich's, 124 
Pappenheim's, 126 
Trichina cystica, 192 



Trichina sanguinis hominis nocturna, 192 

spiralis, 320 
Trichloracetic acid test, 497 
Trichocephalus dispar, 320 
Trichomonads in the feces, 302 
in the sputum, 353 
in the stomach contents, 263 
in the urine, 629 
in vaginal discharges, 667 
Trichomonas vaginalis, 302, 628, 667 
Trichotrachelides, 320 
Triple phosphate crvstals in the sputum, 
363' 
in the urine, 514, 591 
Tripperfiiden, 603 
Trommels test, 517 
Tropaeolin test for hydrochloric acid, 

228 
Trypanosomiasis, 191 
in the blood, 191 
in the cerebrospinal fluid, 655 
Trypsin, 660 

in pancreatic cysts, 660 
test for, 660 
Tube-casts in the urine, 612 
amyloid, 616 
blood, 615 

clinical significance of, 619 
compound hyaline, 615 
epithelial, 615 
fatty, 616 
formation of, 618 
granular, 615 
hyaline, 614 

mode of examination of, 613 
pseudo-, 618 
pus, 615 
staining of, 614 
true, 614 
waxy, 616 
Tubercle bacillus, 354 

cultivation of, 359 
detection of, 354 
in pus, 642 
in the blood, 177 
in the cerebrospinal fluid, 654 
in the feces, 328 
in the milk, 675 
in the mouth, 203 
in the sputum, 354 
in the urine, 626 
Tuberculosis, bacillus of, 354 
Tumor-particles in the gastric contents, 
266 
in the urine, 630 
Typhoid fever, bacillus of, 165, 325 
in the blood, 165 
in the feces, 325 
in the urine, 625 
Tyrosin, 595 

in the sputum, 363 
in the urine, 595 
test for, 597 



694 



INDEX. 



UFFELMANN'S test for lactic acid, 248 
Uncinaria Americana, 317 
dnodenalis, 317 
Unna-Tanzer stain, 352 
Unorganized sediments in urines, 588 
Uraemia, 47 

Urates in urinary sediments, 589 
Urea in the blood, 47 
in the urine, 414 

estimation of, 426 
isolation of, 425 
origin of, 414 
properties of, 422 
tests for, 423 
nitrate, 423 
oxalate, 424 
Ureometers, 426 
Doremus', 428 
Green's, 432 
Hiifner's, 432 
Marshall's, 432 
Simon's, 427 
Squibb' s, 433 
Urethritis, gonorrheal, 644 
Uric acid, 442, 588 

crystals of, 588 
diathesis, 446 
estimation of, 449 

Folin's method, 450 
gravimetric method, 451 
Haycraft's method, 452 
Hopkins' method, 449 
Ludwig - Salkowski's me- 
thod, 453 
in sediments, 588 
in the blood, 48 
in the urine, 442 
properties of, 448 
tests for, 449 
Urinary cylinders, 612 
pigments, 534 
sediments, 585 
Urine, 368 

acetone in, 568 
acidity of, 384 
albumins in, 472 
albumoses in, 486 
alkapton in, 555 
alloxur-bases in, 442 
ammonia in, 439 
amount, 371 
animal gum in, 533 

parasites in, 623 
Bence Jones' albumin in, 487 
benzoic acid in, 457 
bile acids in, 550 

pigments in, 548 
blood in, 545 
carbohydrates in, 508 
casts in, 612 
chemistry of, 385 
chlorides in, 387 



Urine, chromogens in, 537 
crotonic acid in, 576 
chyle in, 579, 600 
color of, 370 
consistence of, 371 
cystin in, 412, 594 
dextrin in, 532 
diacetic acid in, 593 
Ehrlich's diazo-reaction, 559 

benzaldehyde reaction, 564 
epithelium in, 602 
fat in, 578 
fatty acids in, 577 
fecal matter in, 630 
ferments in, 579 
fibrin in, 490 
foreign bodies in, 630 
gases in, 580 
general appearance of, 369 

chemical composition of, 385 
glucose in, 508 
glucuronic acid in, 533 
haemoglobin in, 489 
hippuric acid in, 457 
histon in, 492 

homogentisinic acid in, 555 
indican in, 536 
inosit in, 534 
kreatin in, 460 
kreatinin in, 460 
kryoscopy in, 585 
lactic acid in, 576 
lactose in, 531 
laevulose in, 531 
laiose in, 532 
leucocytes in, 605 
maltose in, 531 
melanin in, 554 

microscopical examination of, 585 
mineral ash, estimation of, 386 
neutral sulphur in, 411 
nitrogen in, 418 
nubecula in, 369 
nucleo-albumin in, 491 
nucleohiston in, 492 
odor of, 371 

organized sediments in, 602 
oxalic acid in, 466 
oxaluric acid in, 466 
oxyamygdalic acid, 577 
oxybutyric acid in, 574 
parasites in, 623 
pentoses in, 532 
peptone in, 489 
phenol in, 555, 566 
phosphates in, 395 
pigments in, 534 

referable to drugs in, 559 
ptomains in, 581 
pus in, 605 
pyrocatechin in, 567 
quantity of, 371 
reaction of, 380 



INDEX. 



695 



Urine, sediments in, 369, 585 

serum-albumin in, 472 

serum-globulin in, 485 

skatoxyl sulphate in, 566 

solids in, 380 

specific gravity of, 375 

spermatozoa in, 622 

sugar in, 508 

sulphates in, 404 

sulphur, neutral, in, 411 

tumor-particles in, 630 

urea in, 414 

uric acid in, 442 

urobilin in, 551 

urochrome in, 535 

uroervthrin in, 536 

urohamiatin in, 543 

uroha?matoporphyrin in, 546 

urorosein in, 544 

vegetable parasites in, 626 

xanthin-bases in, 442, 455 
Urines, blue, 559 

green, 559 
Urinometers, 378 
Urobilin, febrile, 552 

identity with stercobilin, 284 

normal, 535 

pathological, 535, 551 

tests for, 553 

Gerhardt's, 553 
spectroscopic test, 553 
Urobilinogen, 551 
Urobilinuria, 552 
Urochrome, 534 
Uroervthrin, 536 
Urofuscohsematin, 545 
Urohaernatin, 543 
Urohsematoporphyrin, 546 
Uroleucinic acid, 555 
Urophain, Heller's, 544 
Urorosein, 544 
Uroroseinogen, 544 
Urorubroha?matin, 545 
Uroxanthinic acid, 555 
Urrhodinic acid, 555 

VAGIXAL blennorrhea, 668 
discharges, 666 
bacteria in, 667 
during menstruation, 668 
following parturition, 668 
general description of, 666 
in abortion, 670 
in gonorrhoea, 669 
in membranous dvsmenorrhcea, 

669 
in uterine cancer, 669 



Vaginal discharges in vaginitis, 669 

in vulvitis, 669 

parasites in, 669 

reaction of, 667 
Vaginitis exfoliativa, 669 
Vincent's fusiform bacillus, 205 
Vitalli's test for pus, 606 
Vomited material, 259 

bile in, 261 

blood in, 261 

food material in, 259 

mucus in, 260 

odor of, 263 

pancreatic juice in, 261 

parasites in, 263 

pus in, 262 

saliva in, 261 

stercoraceous material in, 262 
Vomitus matutinus, 221, 261 
v. Fleischl's ha?mometer, 150 

WAXG'S estimation of indican, 541 
Wassiliew's estimation of albumin, 
499 
Waxy casts, 616 
Weigert-Ehrlich stain, 358 
Weigert's elastic tissue stain, 351 
Westphal's stain, 136 
Weyl's test for kreatinin, 462 
Whetstone crystals. See Uric acid. 
White blood-corpuscles. See Leucocytes. 
Whooping-cough, bacillus of, 361 
Widal's serum-test, 166 
Williamson's blood-test in diabetes, 46 
Will-Varrentrapp's method of estimating 

nitrogen, 437 
Woodward's method of estimating rnilk- 

proteids, 677 
Worms. See Vermes. 
Wright's coagulometer, 29 
stain, 130 

XAXTHIX-BASES in the blood, 49 
in the feces, 285 
in the urine, 442, 455 
estimation of, 456 
Xanthoproteic reaction, 280 
Xylose in urine, 532 

YEAST-CELLS in the gastric contents, 
264 
in the urine, 628 
Yellow fever, bacillus of, 180 

ZIEHL-XEELSEX stain, 358 
Ziemann's stain, 133 
Zymogens in the gastric juice, 236 



MAR 16 1904 



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